FR2655990A1 - VIRAL AGENT RESPONSIBLE FOR NON-A NON-B HEPATITIS AND POLYPEPTIDES DERIVED THEREFROM FOR USE IN DIAGNOSIS AND VACCINATION. - Google Patents

Abstract

The invention relates to the post-transfusion hepatitis non-A non-B viral polypeptide, the DNA sequences encoding this viral polypeptide, the expression vectors containing these DNA sequences and the hosts transformed by these vectors. expression. The invention also relates to the use of such peptides in diagnostic assays and vaccine formulations.

Description

Viral agent responsible for non-A non-B hepatitis and polypeptides

  derivatives thereof useful for diagnosis and vaccination.

______________

  The present invention relates to separation and

  characterization of the viral agent responsible for the

  non-A non-B post-transfusional patella (PT-NANBH) and in particular to PT-NANBH viral polypeptides, to the DNA sequences encoding these viral polypeptides, to the expression vectors containing these DNA sequences, and to the cells hosts transformed by these vectors

  of expression It also refers to the use of

  of a DNA sequence in a nucleic acid hybridization assay for the diagnosis of PT-NAN Bi H.

  It also relates to the use of polypeptides

  PT-NANBH viral or polyclonal antibodies or

  against these polypeptides in an immunological

  analysis for the diagnosis of PT-NANBH or in a vac-

for his prevention.

  Non-A, non-B hepatitis (NANBH) is by definition an exclusion diagnosis and has generally been used to describe cases of viral hepatitis

  the human being who are not due to hepatitis viruses

  In most cases, the cause of infection has not been identified, although for clinical and epidemiological reasons it has been

  a number of agents who could be responsible for

  sables, see Shih et al (Proq Liver Dis, 1986, 8, 433-452) In the United States alone, up to% of blood transfusions may have a NANBH

  as a consequence, making it an important problem.

  Even for PT-NANBH, there may be at least several viral agents responsible for the infection and

  over the years, the claims of identity

  tification of the agent did not fail, but no

has not been established.

  European patent application 88310922 5 tends to describe

  the separation and characterization of the

  responsible for PT-NANBH, which is called

  also hepatitis C virus (HCV) Uine biblio-

  library of AD Nc was prepared from nucleic acid

  Viral obtained from a chimpanzee PT-NANBH and was selected using human antisera Uine series of clones were isolated and sequenced Nucleic acid and amino acid sequence data

  resulting from it and which are described in the application,

  present approximately 70% of the 10 kb viral genome

  and are entirely derived from its 3 'end cor-

  corresponding to the non-structural programming region

  SOE. The inventors have now isolated and characterized the viral polypeptides of PT-NANBH by cloning.

  and the expression of DNA sequences encoding these poly-

  viral peptides Surprisingly, the data

  born of nucleic acid and amino acid sequence

  have considerable differences with the data

  corresponding figures reported in the application for

  European patent 88310922 5 On the whole, these

  ferences are about 20% at the level of the nu-

  15% at the amino acid level, but some regions of the sequences even show

  larger differences The overall level of differences

  is more important than we would expect

  for two isolates of the same virus, even taking into account

  geographical factors, and can be explained by

one of two reasons.

  First, the inventors of the present invention and those of the aforementioned European patent application

  used different sources for the nu-

  key used in the cloning of c DNA In particular

  linking the European patent application describes the use of

  chimpanzee plasma as a source of the starting material for the viral nucleic acid, the virus having

  been transmitted to a chimpanzee on two occasions.

  NANBH is, of course, a human disease and the passage of the virus into a foreign host, even if it is closely related to humans, is likely to result in an intensive mutation of the viral nucleic acid.

  this is the sequence data contained in the application

  of European patent 8831022 5 may not be

  exactly representative of the actual viral agent

  In contrast, the inventors of the present invention have used viral nucleic acid from a source of human plasma as starting material for the cloning of cDNA.

  sequence data thus obtained are more likely

  to correspond to the native sequences of nu-

  key and amino acid of PT-NANBH.

  Secondly, it can happen that the viral agent

  in the form of more than one subtype and that the sequence data described in the European patent application and those elucidated by the inventors herein correspond to separate and distinct subtypes of the same viral agent Alternatively it may to be done that the level of difference between the two

  groups of sequence data either d to a combination

of these two factors.

  The present invention provides a PT-4 viral polypeptide

  NANBH comprising an antigen having an acid sequence

-3-

  amine, which is at least 90% homologous to the

  amino acid number indicated in SEQ ID NOS: 314,18,19,20,21 or 22, or an antigenic fragment thereof. SEQ ID Nos. 3,4,5,18,19,20,21 or 22 indicate the sequence of

  the amount of amino acid as deduced from the

  Nucleic acid sequence The amino acid sequence will preferably be at least 95% or even 98% homologous with the amino acid sequence indicated in SEQ ID NO 3,4,5,18,19,20,21 or 22 Optionally antigen

  can be fused with a heterologous polypeptide.

  Two or more antigens may optionally be used in combination or fused to a single polypeptide. The use of two or more antigens in this manner in a diagnostic assay provides more reliable results than the use of the blood screening assay. for the PT-NANBH virus We will get

  preferably an antigen in the program region.

  (the 5 'end) and another in the unstructured programming region (I end

  3 ') Preference is given in particular to the

  antigens with each other to give a polyp.

  tide resulting from recombination.

  A series of advantages can be achieved by having the individual antigens combined in a fixed ratio determined in advance (usually equimolar) and not producing, purifying and

  characterize only one polypeptide.

  An antigenic fragment of an antigen having a

  at least 90% homologous amino acid with that indicated in SEQ ID NO: 3,4,5,18,19,20,21 or 22 will preferably contain a minimum of five, six, seven, eight, nine or ten, fifteen, twenty, thirty, forty or fifty amino acids The antigenic locations of these antigens can be identified

  using standard procedures. These may imply

  fragmentation of the polypeptide itself by means of proteolytic enzymes or chemical agents and subsequent determination of the ability of each fragment to bind to the antibodies or to elicit an immune response when inoculated with an animal or

  in an appropriate in vitro model system (Stroh-

  maier et al., J Gen Virol, 1982, 59, 205-306).

  Alternatively, the DNA encoding the polypeptide may be fragmented by digestion with reducing enzymes or other reputable techniques and then introduced into an expression system to produce fragments (optionally fused to a polypeptide generally of bacterial origin). The resulting results are evaluated as previously described (Spence et al., J Gen Virol, 1989, 70, 2843-51, Smith et al.

  1984, 29, 263-9) Another approach is to

  chemically synthesize short peptide fragments (length of 3-20 amino acids, conventionally 6 amino acids long) which cover the entire long polypeptide sequence with each peptide overlapping the adjacent peptide (This overlap can be from I- 10 amino acids, but the ideal

  is n-i amino acids, where N is the length of the pepti-

  of; Geysen et al, Proc Natl Acad Sc, 1984, 81,

  3998-4002) Each peptide is then evaluated as de-

  previously written, except that it is usually first coupled to a carrier molecule to facilitate the induction of an immune response Finally there are prediction methods that include the analysis of the

  sequence from the point of view of particular characteristics

  such as hydrophilicity, which is thought to be

  that they relate to important locations

  Immunologically speaking (Hopp and Woods, Proc Natl Acad Sc, 1981, 78, 3824-8, Berzofsky, Science, 1985, 229, 932-40) These predictions can then be tested by the polypeptide or peptide approaches.

  resulting from the recombination previously described.

  The viral polypeptide will preferably be provided in a pure form, ie with a purity higher than

%, even 95%.

  The PT-NANBH viral polypeptide of the present invention may be obtained by means of an amino acid synthesizer, if it is an antigen having not more than about thirty residues or by the

  the DNA resulting from the recombination.

  The present invention also provides a DNA sequence encoding a PT-NANBH viral polypeptide as

defined here.

  Your DNA sequence of the present invention can be

  synthetic or cloned The DNA sequence will be

  as indicated in SEQ ID NO: 3,4,5,18,

19.20.2 l or 22.

  To obtain a PT-NANBH viral polypeptide comprising multiple antigens, it is preferable to melt the individual programming sequences into a single open reading frame.

  to be carried out in such a way that the antigenic activity

  that each antigen is not compromised by

  important by its position in relation to a

  antigen will be granted, of course,

-5-

  particular to the nature of the sequences in the

  right of the junction between the antigens The methods of obtaining such simple polypeptides are well

known in this field.

  The present invention also provides an expression vector containing a DNA sequence such that

  defined here, and capable, in the presence of an appropriate host

  requested, to express the DNA sequence to produce a

viral polypeptide PT-NANBH.

  The expression vector normally contains elements

  DNA control elements that express

  of the DNA sequence in a suitable host

  may vary depending on the host, but

  usually take an activator, a link site

  ribosome sound, start and stop sites

  transcriptional site, and a transcription stop site.

  Examples of such vectors are plasmids and viruses. Expression vectors of the present invention.

  invention include both extra-vector

  chromosomes and vectors that are integrated into the chromosome of the host cell For use in E. coli, the expression vector may contain

  the DNA sequence of the present invention optionally

  as the 5 'or 3' end of the encoding DNA sequence, e.g., β-galactosidase or at the 3 'end of the sequence

  DNA encoding, for example, the trp E gene.

  in the insect bacillovirus system (Ac NPV), the DNA sequence is optionally

  with the programming sequence of polyhedrin.

  The present invention also provides a host cell transformed with an expression vector such as

defined i ci.

  Examples of host cells used with the pre-

  invention include prokaryotic cells

  and eukaryotic, such as bacterial cells

  natives, yeast, mammals and insects.

  peculiarities of such cells are E coli,

  S cerevisiae, P pastoris, ovarian

  Chinese hamster and mouse, and Spodoptera

  fruqiperda and Tricoplusia ni The choice of cellulose

  the host may depend on a variety of factors, but whether the post-translational modification of the polypeptide

  Viral PT-NANBH is important, we will choose

a eukaryotic host.

  The present invention also provides a method of

  to prepare the PT-NANBH viral polypeptide which

  cloning or synthesizing a DNA sequence encoding the PT-NANBH viral polypeptide, as defined herein, inserting the DNA sequence into a vector

  of expression capable, in a suitable host, of

  primer, to transform a host cell with the vec-

  expression, to cultivate the cell

  host transformed and isolate the viral polypeptide.

  Cloning of the DNA sequence can be performed aul

  standard procedures well known in this field.

  Moreover, it is particularly advantageous in

  these procedures to use the sequence data re-

  here to facilitate identification and

  separation of the desired cloned DNA sequences.

  The RNA will preferably be isolated by pelletizing the

  rus from the plasma of infected humans identified

  by implication in the transmission of PT-NANBH.

  The isolated RNA is subject to reverse transcriptase

  in the selective priming NCD or by oligo-d T Fa-

  culturally the RNA can be subjected to a step of

  pre-treatment to remove any secondary structure

  which could interfere with AD Nc synthesis, for example, by heating or reacting with

  Methyl mercuric hydroxide The AD Nc is usually

  It is then modified by addition of linkers followed by digestion with a reducing enzyme. It is then inserted into a cloning vector, such as BR 322 or a derivative thereof or lambda gt1 O and gtll vectors (Huynh et al. , DNA Cloning, 1985, Vol 1: A Practical Approach, Oxford, IRC Press), packaged in

  virions where appropriate, and the resultant DNA molecules

  both of the recombination used to transform

  E coli and thus generate the desired library.

  The library can be examined selectively

  using a typical selection strategy If it's an expression library, it can be

  selectively examined by means of an immunological

  logic with antisera obtained from the same

  me source of plasma as the starting material for RNA and also with antisera from additional human sources believed to be positive for

  anti-PT NANBH antibodies.

  human serum usually contains anti-

  body against E coli that can give rise to a large clean movement during selection, it is best to treat them first with a lysate

  untransformed E. coli so as to remove these

  It is advantageous to use a negative control with antisera from accredited human donors, ie human donors who have been repeatedly tested and do not have antibodies against viral hepatitis. A strategy of

  alternative selection would be to use one or more

  oligonucleotides labeled as hybridization probes The use of oligonucleotides in

  I selectively examine a library c DNA is usually

  easier and more reliable than the selection

  with anti sera Oligonucleotides will be syn-

  preferably, using the information of the DNA sequence contained herein. One or more additional screening rounds of one kind or the other can be performed to characterize and

identify positive clones.

  After identifying a first positive clone, the bi-

  library may be reconsidered selectively to

  detect additional positive clones using

  the first clone as a hybridization probe Al-

  other or additional, other libraries may

  can be prepared and these can be selected

  immuno-selectors or probes

  In this way, one can obtain from

very DNA sequences.

  Alternatively, the DNA sequence encoding the poly-

  PT-NANBH viral peptide can be synthesized using

  standard procedures and this solution may be preferable

  DNA cloning in certain circumstances

  (Gait Oligonucleotide Synthesis: A Practical AP-

proach, 1984, Oxford, IRL Press).

  So cloned or synthesized, the desired DNA sequence

  It can be inserted into an expression vector using known standard techniques. The expression vector is normally cleaved by means of reducing enzymes and the inserted DNA sequence by blunt-ended or zigzag ligation. cleavage is usually performed at a restriction site

  in an appropriate position in the expression vector

  so that once inserted, the DNA sequence is under the control of the functional elements

  of DNA that realize its expression.

  The transformation of a host cell can be effected

  killed by standard techniques A phenotypic marker is usually used to distinguish between transformants that have successfully resumed the expression vector and others that are cultured.

  of the transformed host cell and the separation of the

  The PT-NANBH viral polypeptide can also be

typical techniques.

  8 - The specific antibody of a PT-NANBH viral polypeptide of the present invention can be obtained by means of the

  polypeptide It may be polyclonal or monoclonal.

  The antibody can be used for

  quality control of batches of PT-NANBH viral polypeptides; the purification of a PT-NANBH viral polypeptide

  or viral lysate; the topography of epitopes; lors-

  that it is labeled as a conjugate element in a competitive standard assay for the detection of antibodies; and

  in antigen detection assays.

  The polyclonal antibody against a PT-viral polypeptide

  NANBH of the present invention can be obtained in

  injecting a PT-NANBH viral polypeptide, optionally

  coupled with a support to promote an immune response

  in a mammalian cell: mouse, rat, sheep

  or rabbit, and recovering the antibody thus produced.

  The PT-NANBH viral polypeptide is usually administered

  in the form of an injectable formulation in

  which polypeptide is mixed with a physiological diluent

  siologically acceptable Adjuvants such as

  Freund's complete adjuvant (FCA) or adjuvant

  Complete Freund (FIA) can be included in the formulation The formulation is normally injected into the host over an appropriate period of time, with plasma samples being taken at intervals

  regular tests for testing viral antibodies

  When an appropriate level of activity is achieved, the host leaks. The antibody is then extracted from the blood plasma and purified by standard procedures, by

  protein A or by

ion exchange resin.

  The monoclonal antibody against a PT-NANBH viral polypeptide of the present invention can be obtained by

  merging cells from an immortal cell line

  lysing cells producing antibodies against the viral polypeptide and culturing the immortal fused cell line. Typically, a non-mammalian hast is inoculated.

  human, such as mouse or rat, the viral polypeptide.

  After sufficient time for the host to

As an antibody response, antibody-producing cells, such as splenocytes, are removed. The cells of an immortalizing cell line, such as a mouse or rat myeloma cell line, are fused with the antibody-producing cells. the resulting mergers examined from

  selectively to identify a cell line

  re, such as a hydridome, secreting the monocular anticorns

  The fused cell line can be cultured and the purified monoclonal antibody of the

  culture medium in a manner similar to purifying

polyclonal antibody.

  Diagnostic tests based on the present invention

  can be used to determine the presence or absence of PT-NANBH infection. They can also be used to

  this infection, for example in

terféron.

  In an attempt to diagnose an infection

  There are basically three distinct approaches to

  which may be adopted involving the detection of

  viral nucleic acid, viral antigen or

  viral ticbody The viral nucleic acid is generally

  regarded as the best indicator of the pre-

  of the virus itself and identify substances

  these likely to be infectious Moreover, the detection of nucleic acid is generally not

  as direct as the detection of antigens or anti-

  body since the target level can be very low.

  The viral antigen is used as a marker for the

  presence of the virus and as an indicator of

  Depending on the virus, the amount of antigen present in a sample may be very small and

  difficult to detect The detection of antibodies is

  directly because, in fact, the immune system of

  the host amplifies the response to infection with

  high amounts of antibodies that circulate

  The nature of the antibody response can often be

  may be clinically useful, eg

  Ig M body rather than IgG indicate a

  recent infection, or the response to a particular viral antigen associated with virus clearance

  the exact approach adopted for the diagnosis of a

  viral fection depends on the particular circumstances

  and information sought In the case of the PT-

  NANRH, a diagnostic analysis may incorporate any

  which of these three approaches.

  In a test for the diagnosis of PT-NANBH impli-

  as for the detection of viral nucleic acid, the method may comprise the hybridization of the viral RNA present in a sample or the AD Nc synthesized from

  of this viral RNA, with a corresponding DNA sequence

  to the nucleotide sequence of SEQ ID No. 3,4, 8,19,20,21 or 22 and the selection of the resulting nucleic acid hybrids to identify any acid

  viral nucleic acid PT-NANBH The application of this

  thode is usually limited to a sample of one

  appropriate tissue, such as liver biopsy, or RNA vi

  ral is likely to be present in strong

-

  portions The DNA sequence corresponding to the sequence

  nucleotide of SEQ ID No. 3,4,5,18,19, 20,21 or 22 may take the form of an oligonucleotide or a

  AD Nc sequence optionally containing a plasmid.

  The selection of the nucleic acid hybrids will preferably be carried out by means of a DNA sequence

  marked One or more additional selection rounds

  of one type or the other can be carried out in order to better characterize the hybrid and thus identify any PTNANBH viral nucleic acid.

  hybridization and selection are carried out

known procedures.

  As a result of the limited application of this method in the viral nucleic acid assay, a preferred and more practical method involves the synthesis of

  AD Nc from the viral RNA present in a sample of

  the amplification of a preselected DNA sequence

  corresponding to a subsequence of the sequence

  nucleotide sequence of SEQ ID No. 3,4,5,18,19,20,21

  or 22, and the identification of the DNA sequence

  The test sample may be taken from any suitable tissue or physiological fluid and will preferably be concentrated for all the viral RNA present. An example of a suitable tissue is a biopsy.

  Examples of physiological fluid suitable for

  are: urine, plasma, blood, serum,

  sperm, tears, saliva or cerebrospinal fluid

  The first choice examples are serum and plasma. The synthesis of AD Nc is normally carried out by

  reverse transcription primed with separate primers

  lective, defined or oligo-d T The most advanced primer

  tageuse is an oligonucleotide corresponding to the sequence

  nucleotide sequence of SEQ ID No. 3,4,5,18,19,20,21 or 22 and designed to enrich in AD Nc containing the preselected sequence. The amplification of the preselected DNA sequence is preferably performed by means of a polymerase chain reaction (PCR) technique (Saiki et al., Science, 1985, 230, 1350-4). a pair of oligonucleotide primers whose

  one corresponds to a portion of the sequence of nu-

  cleotide of SEQ ID No. 3,4,5,18,19,20,21 or 22 and

  the other is on the 3 'side of the first and corresponds to

  to a portion of the complementary sequence, the

  pair defining between them the DNA sequence

  Oligonucleotides generally have a length of at least 15, optimally from 20 to 26 it -

  bases and although some errors may be tolerated

  by varying the conditions of the reaction, the ex-

  tremity 3 'of the oligonucleotides should be perfect-

  The distance between the 3 'ends of the oligonucleotides can be from about 100 to about 2000 bases. Practically one of the oligonucleotides of the pair used in this technique is also used to initiate the synthesis of oligonucleotides. CDNA The PCR technique itself is performed on AD Nc as a single chain using an enzyme such as Taq polymerase, and a surplus of oligonucleotide primers.

  more than 20-40 cycles according to public protocols

  (Saiki et al., Science, 1988, 239, 487-491).

  To refine the technique, there may be several rounds of amplification, each round being initiated by another pair of oligonucleotides.

  first round of amplification, an internal pair of oli-

  gonucleotides defining a shorter DNA sequence (say, 50 to 500 bases) can be used

  for a second round of amplification In this refining

  somewhat more reliable, which is referred to as "Nested ACP", it is of course the

  final amplified DNA sequence which constitutes the

  preselected quence (Kemp et al, Proc Natl Acad Sc, 1989, 86 (7), 2423-7 and Mullis et al, Methods in

Enzymoloqy, 1987, 155, 335-350).

  The identification of the preselected DNA sequence can be done by analysis of the PCR products on an agarose gel The presence of a molecular weight band

  calculated for the preselected sequence is an indi-

  positive cation of the presence of viral nucleic acid

  in the sample Alternative methods of identifying

  include those based on the transfer

  Southern, the transfer of points, the restriction of

  gomer and sequencing of DNA.

  The present invention also provides a test kit for the detection of PT-NANBH viral nucleic acid, which comprises i) a pair of oligonucleotide primers, one of which

  corresponds to a portion of the nucleotide sequence

  otides of SEQ ID No. 3,4,5,18,19,20,21 or 22 and the other of which is on the 3 'side of the first

  and corresponds to a portion of the sequence

  complementarily, the pair defining between them a preselected DNA sequence; 12 -

  ii) a reverse transcriptase enzyme for the syn-

  AD Nc thesis from the RNA of the sample

  lon upstream of the primer corresponding to the se-

  nucleotide sequence complementary to SEQ ID NO: 3,4,5,18,19,20,21 or 22; iii) an enzyme capable of amplifying the preselected DNA sequence; and optionally

  iv) washing solutions and reaction buffers.

  Advantageously, the test kit also contains a

  positive control sample to facilitate the identification

  of the viral nucleic acid.

  The characteristics of the primers and enzymes will preferably be those indicated above in relation to

with the ACP technique.

  In a test for the diagnosis of PT-NANBH impli -

  when detecting viral antigens or viral antibodies, the method may comprise contacting a sample with a PT-NANBH viral polypeptide of the present invention or a polyclonal or monoclonal antibody against that polypeptide, and determining

  the existence or not of an antigen-anti-

  body contained in the sample The sample may be taken from any suitable tissue or physiological fluid mentioned above for the detection of

  viral nucleic acid If we obtain a physiological fluid

  ifological, it could be optimally

  male to detect any antigen or viral antibody.

present.

  A series of analysis formats can be used.

  The PT-NANBH viral polypeptide can be used to

  capture the antibodies selectively

  port to PT-NANBH in the solution, to selectively label the antibodies already captured or both to capture and label the antibodies. Additionally, the viral polypeptide can be used in a variety of homogeneous assay formats in which the antibody reacting with the antigen is detected

  in the solution without phase separation.

  The types of tests in which the polyp

  PT-NANBH viral tide to capture the antibodies in the solution involve the immobilization of the polypeptide on a solid surface This surface should be able to be washed in one way or another Examples of suitable surfaces are polymers of different types (molded in microtiter wells, beads, pipettes of different types, pipettes 13 -

  Pasteur; electrodes; and optical devices

  particles) (eg latex, erythrocytes

  stabilized; bacterial or fungal cells; spo-

  res; containing soils containing gold or other

  rate; and protein gels), the usual particle size being from 0.02 to 5 microns, membranes (eg, nitrocellulose, paper, acetocellulose;

  and high porosity / high surface membranes of a

organic or inorganic material).

  The attachment of the PT-NANBH viral polypeptide to the surface can be by passive adsorption of an optimal composition solution containing surfactants,

  solvents, salts and / or chaotropes; or by

  its active chemical The active connection can be ef-

  made by a variety of reactive or activatable functional groups that may be exposed on the surface (eg condensing agents;

  active acids, halides and anhydrides; groups

  oxhydrile or carboxyl-groups sulfydrylef grou-

  pes carbonyl; diazo groups; or unsaturated groups).

  Optionally the active connection can be done via

  a protein (itself attached to the passive surface-

  or active binding), such as albumin or casein, to which the viral polypeptide may be bound

  chemically by a whole series of methods.

  Using a protein in this way can

  confer advantages as a result of an isolated point

  cation, charge, hydrophilicity or any other physicochemical property The viral polypeptide

  can also be attached to the surface (general-

  not necessarily a membrane) following

  electrophoretic separation of a reaction mixture

  such as immune precipitation.

  After contacting (having reacted) the sur-

  the PT-NANBH viral polypeptide-bearing side with a sample, allowing sufficient time for reaction, and where appropriate, removing excess sample by one of several means

  available (washing, centrifugation, filtration,

  (eg, gnatism or capillary action), the captured antibody is detected by any means that will give a detectable signal. For example, it can be achieved by using a labeled molecule or particle as indicated above that will react with the antibody. capture

  (eg, protein A, protein G, etc.;

  this or subtype of anti-immunoglobulin; rheumatic factor

  toïde; or antigen of the antigen used competitively or blocking), or any molecule containing

  an epitope contained in the polypeptide.

  14 - The detectable signal can be optical, radioactive or physicochemical and can be supplied directly

  marking the molecule or particle with, for example

  ple, a dye, a radioactive label, an electroactive species, a magnetically resonant or fluorogenic species, or indirectly by marking the molecule or particle by means of an enzyme capable of giving rise to a measurable change of any type Alternatively, the detectable signal can be obtained, for example, by agglutination or by a diffraction or birefringence effect if the surface

is in the form of particles.

  Assays in which a PT-NANRH viral polypeptide is used itself to label an already captured antibody require some form of labeling of the antigen that will allow its detection.

  can be direct by attaching chemically or passively

  for example, a radioactive label, a magnetically resonant species, a marker in the form of a particle or enzyme to the polypeptide; or indirect

  attaching any form of brand to a molecule that

  will itself act with the polypeptide The brand-binding chemistry to the PT-NANBH viral polypeptide can be done directly via a half already present in the polypeptide, such as an amino group, or by

  through an intermediate half, such as a group

  The capture of the antibody can be

  on any of the surfaces already mentioned.

  by any reagent including ad-

  passive or activated sorption that will result in the

  binding of specific complexes of antibodies or

  In particular, the capture of the antibody

  would be done by an anti-species or a subtype

  of anti-immunoglobulin, by rheumatoid factor, protein

  A, G, etc., or any molecule containing an epi

tope contained in the polypeptide.

  The labeled PT-NANBH polypeptide can be used in

  a competitive bond or the binding to any molecule

  the specific on any of the surfaces

  as an example above is blocked by the anti-

  Alternatively, it can be used in a non-competitive manner in which the antigen in the sample is specifically or non-specifically bound to one of the above surfaces and is also bound to a specific molecule. bi or

  versatile (eg, an antibody), its other

  these being used to capture the polypeptide

  than. Often, in homogeneous assays, the PTNANBH viral polypeptide and an antibody are labeled separately so that when the antibody reacts with the viral polypeptide in free solution, the two tags interact to allow, for example, the transfer of non-radiating energy captured by a mark towards the other mark with appropriate detection of the second excited mark or the first mark

  extinguished (eg by fluorimetry, magnetic resonance

  that or measurement of enzymes) The addition of viral polypeptide or antibody in a sample is reflected

  by a restriction of the interaction of the mar-

  and therefore by a different level of the signal in the detector.

  A suitable test format for the detection of

  PT-NANBH is the format of the immunoassay with

  sandwich enzyme (EIA) A viral polypeptide PT-

  NANBH is applied to microtiter wells A sample and a PT-NANBH viral

  are coupled an enzyme, are added simultaneously.

  Any PT-NANBH antibody present in the sample binds with the virus polypeptide

  cup and with the viral polypeptide coupled to the enzyme.

  Typically the same viral polypeptide is used

  on both sides of the sandwich After washing, the enzyme

  Bindery is detected by means of a specific substrate involving a color change. A test kit for use in such an EIA comprises: (1) an enzyme labeled PT-NANBH viral polypeptide; (2) a substrate for the enzyme; (3) means providing a surface on which a PT-NANBH polypeptide is immobilized; and (4) optionally, washing solutions and / or buffers.

  The viral polypeptides of the present invention can

  be incorporated into a vaccine formulation

  to induce immunity against PT-NANBH in homosexual

  To this end, the viral polypeptide may be

  in association with an acceptable carrier of

pharmaceutical point of view.

  For its use in a vaccine formulation,

  the viral polypeptide may optionally be presented

  as an element of a particle born of fusion

  of the hepatitis B nucleus, as described in

  ke et al (Nature, 1987, 330, 381-384), or a polylysine-based polymer, as described in Tam (PNAS, 1988, 85, 5409-5413). Alternatively, the polypeptide 16-

  viral can optionally be attached to a

  particulate matter such as liposomes or ISCOMS.

  Acceptable carriers from the pharmaceutical point of view

  Among other things, these are liquid media that can be used to convey the viral polypeptide in the body of a patient. An example of such liquid media is saline. The viral polypeptide itself can be dissolved or suspended in suspension.

solid form in the support.

  The vaccine formulation may also contain a

  adjuvant to stimulate the immune response and thus, ac-

  Percentage of vaccine effect Examples of adjuvants are, among others: aluminum hydroxide and

aluminum phosphate.

  The vaccine formulation may contain a concentration

  final concentration of the viral polypeptide ranging from 0.01 to 5 mg / ml, preferably from 0.03 to 2 mg / ml. The formulation

  vaccine can be incorporated in a sterile container.

  which is then sealed and stored at low temperatures

  e, for example, at 40 ° C, or can be lyophilized.

  To introduce immunity against PT-NANBH in humans, one or more doses of the vaccine formulation may be administered. Each dose may be from 0.1 to 2 ml, preferably from 0.2 to 1 ml. to induce immunity against PT-NANBH in humans

  involves administering an effective amount of

  the vaccine as previously stated.

  The present invention also provides the use of a PT-NANBH viral polypeptide in the preparation

  of a vaccine for use in the induction of

  munity against PT-NANBH in humans.

  The vaccines of the present invention may be

  by any method appropriate to the administration

  of vaccines, including oral and

  (eg, intravenous, subcutaneous or intravenous).

  Muscle) Treatment may consist of a single dose of vaccine or in divided doses over time. The following transformed E. coli strains were

  deposited with the National Collection of Cultiv-

  (NCTC), Central Public Health Laboratory, 61,

  Colinda I Avenue, London, NW 9 5 HT on the dates indicated

  : 17 - i) E coli TG 1 transformed with p DX 113 (WDOO 1); Deposit

n NCTC 12369; December 7, 1989.

  ii) E. coli TG 1 transformed with p DX 128 (WD 002); Deposit

n NCTC 12382; February 23, 1990.

  iii) E coli TG 1 transformed with p 136/155 (WD 003); Of-

  PO NCTC 12428; November 28, 1990.

  (iv) E. coli TG 1 transformed with p 156/92 (WD 004):

* deposit NCTC 12429; November 28, 1990.

  (v) E coli TG 1 transformed with p 129/164 (WD 005); Of-

  deposit N O NCTC 12430; November 28, 1990.

  (vi) E. coli TG1 transformed with p DXI 36 (WD 006); Deposit

n NCTC 12431; November 28, 1990.

  In the figures, Figure 1 shows the production

  of DX 122 described in Example 7, FIG.

  the two alternative melts described in Example 17, and FIG.

restriction, of SEQ ID Nos. 21 and 22.

  In the sequence list, there are SEQ ID lists

  No 1 to 25 to which reference is made in the

  and in the claims.

  The following examples serve to illustrate the invention.

EXAMPLE 1 Synthesis of AD Nc

  The pooled plasma (160 mls) of two people (called

  A and L) known to have transfected NANBH via transfusions was diluted (1: 2.5) with a solution

  phosphate-buffered saline (PBS) and then

  leaked at 190,000 g (eg, 30,000 rpm in an 8 × 50 MSE rotor) for 5 hours at 4 ° C. Supernatant was retained as a source of specific antibodies for subsequent selection of AD Nc I libraries. The pellet was resuspended in 2 ml of 20 m M trichlorohydrate, 2 mM EDTA 3% SDS, 0.2 M NaCl (2 x PK) extracted 3 times with an equal volume of phenol, 3 times with 10 ml. chloroform, once with ether, and

  then precipitated with 2.5 volumes of ethanol at -20 ° C.

  The precipitate was resuspended in 10 μl of

  10 mM tris-hydrochloride, 1 m M EDTA at pH 8.0 (TE).

  The nucleic acid was used as a template in an AD Nc synthesis kit (Amersham International plc,

  Amersham, United Kingdom) with oligo-d T priming and hexa-

  selective nucleotide The reaction conditions were as recommended by the supplier of the 18 -

  Specifically, lul of nucleic acid was used.

  se for a first strand synthesis reaction which

  has been labeled (4 _-32 P) d CTP (Amersham;

  3000 Ci / mmol) in a final volume of 20 μl and incubated at 42 ° C. for 1 hour.

  strand reaction was then used for the second

  twelfth synthesis reaction of the strand, containing E co-

  R Nase H (0.8 U) and DNA polymerase I (23 U) in a final volume of 100 μl, incubated at 12 ° C for

  60 minutes, then at 22 C for 60 minutes

  The reaction appears to have been incubated at 700 C

  After 10 minutes, placed on ice, 1 U of T 4 DNA polymerase was added and then incubated at 37 ° C for 10 minutes.

adding 5 μl of 0.2 M EDTA p H 8.

  Unincorporated nucleotides were removed in step

  the reaction in a NICK column (Pharmacia Ltd,

  Milton Keynes, United Kingdom) The AD Nc was then ex-

  run twice with phenol, three times with chloroform, once with ether and then 20 μg of dextran was added before precipitation with

2.5 volumes of 100% ethanol.

  EXAMPLE 2 Production of expression libraries The dried AD Nc pellet was resuspended in 5 μl of sterile TE and then incubated with 500 ng of Eco RI linkers (Pharmacia, GGATTCC phosphorylated) and

  0.5 U of T 4 DNA Jigase (New England Bio Labs, Beverley

  ley, MA, USA) in a final volume of 10 μl containing m M tris-HCl p H 7.5, 10 m M Mg C 12, 10 m M DTT, 1 m M

  ATP for 3 hours at 15 ° C. The ligase was neutralized

  on heating at 65 ° C for 10 minutes and AD Nc was digested with 180 U of Eco RI (BCL, Lewes, UIJ K) in a final volume of 100 μl at 37 ° C for 1 hour. was added to a final concentration of 10 mM and the entire reaction loaded on an Ac A 34 (LKB) column. Fractions (50 μl) were collected and counted. The peak of AD Nc in the excluded volume (980 cpm) was pooled, extracted twice with phenol,

  three times with chloroform, once with

  ther and then precipitated with ethanol.

  AD Nc ds was resuspended in 5 μl of TE and

  ligated on arms lambda gtll Eco RI (Gibco, Poland).

  ley, Scotland) in an ul reaction containing 0.5 U T 4 DNA ligase, 66 m M tris-hydrochlorate, 10 m M

  Mg C 12, 35mM DTT p H 7.6 at 15C overnight.

  After neutralization of the ligase by heating at 65 ° C. for 10 minutes, 5 μl of the reaction was added to an Amersham refolding reaction and incubated at 19 ° C. for 2 hours. The folded material was titrated

  on the strand Y 1090 E coli (Huynh et al 1985) and contains

  a total of 2.6 x 104 resulting from the recombin-

his.

  Monolayer cells (Y 0190) were prepared

  10 ml of L-broth was inoculated with a single colony of agar plate and shaken overnight at 37 ° C. The next day, 0.5 ml of the overnight culture was diluted with 10 ml of Fresh L-broth and 0.1 ml IM Mg SO 4 and 0.1 ml 20% (w / v) maltose were added. The culture was stirred for 2 hours at 37 ° C., the bacteria harvested by centrifugation at 5.000 g for 10 minutes. minutes and resuspended in 5 ml Om M Mg SO 4 to produce the stock of

  cells for monolayers A portion (lul) of the material

  folded was mixed with 0.2 ml of monolayer cells, incubated at 37 ° C for 20 minutes before adding 3 μl of cover agar and the whole mixture poured onto a 90-min L-agar plate After incubation overnight at 47 C, the plates were counted and the total number of phages resulting from recombination was determined. The folded material

  remaining (500 μl) was stored at 4 ° C.

  Additional libraries have been prepared for

  substantially similar way.

  EXAMPLE 3 Selection of Expression Libraries The initial library described in Example 2 was

  plated on yarn Y 1090 E coli at a density of about

  5 x 103 pfu per 140 mm plate and incubated at 37 ° C

  for 2 hours until the plates are visible

  sterile nitrocellulose filters impregnated with IPTG (isopropyl-thiogalactoside) were left in contact with the plate for 3 hours

  and then removed The filters were first

  by incubation with blocking solution (3% (w / v) BSA / TBSTwee (10 mM Tris-HCl p H 8, 150 mM NaCl, 0.05% (v / v) Tween 20) containing 0 , 05% bronidox) (20 mls / filter) and then transferred to the buffer solution

  binding (1% (w / v) BSA / TBS / Tween containing 0.05% bro-

  nidox) containing purified antibodies (by chromato-

  Ion exchange resin graphy) from pooled A & L plasma (20 μg / ml). After incubation at room temperature for 2 hours, the filters were washed three times with TBS-Tween and then incubated in the binding buffer solution. containing an anti-human serum of biotinylated sheep (1: 250) After 1 hour at room temperature, the filters were washed 3

  twice with TBS / Tween and then incubated in the

  binding buffer containing a streptococcal

  - tavidin / peroxidase (1: 100) Signal developed with DAB Positive signals appeared in form

of plates (colored).

  Out of a total of 2.6 x 104 selected plates, 8 positive signals were obtained in the first round of selection Using the filters as the template, the regions of the original plates corresponding to these

  positive signals were picked by means of a

  Sterile Pasteur Pette The agar plugs have been

  suspended in 0.1 ml of SM buffer and the phage

  The phage titer of each cap was determined on the strand Y 1090 E. coli

  phage stock of each cork was then re-examined

  Selectively mined as before on separate 90 mm plates at a density of about 1 x 103 pfu per plate Of the 8 positive signals of the first round, one was clearly positive in the second round, ie> 1% plates positive it was called JG 2 This corresponds to a positive rate of 40/106

in the library.

  This and other positive phages similarly identified in other AD Nc libraries described in Example 2 were then purified by repeated rounds of selective examination of low density plates (I-200 pfu / plate 90 mm) until 100% of the plates are positive with the selection of antibodies A & L Three phages resulting from this recombination

are JGI, JG 2 and JG 3.

  EXAMPLE 4 Secondary Selection of JG 1, JG 2 and JG 3 with Serum Sampling Each of the phages resulting from recombination, JG 1, JG 2 and JG 3, were plaque purified and stored as titrated stocks in a buffer. MS at 40 C. These phages were mixed (1: 1) with a pool of phages identified as negative in Example 3 and the mixture used to contaminate Y strand 1090

  E. coli at 1000 pfu per plate Plant detachments

  that were carried out and treated as described in the Ex-

  Example 3, except that the filters were divided into quadrants and each quadrant was incubated with a different antibody; these were A & L antibodies (20 μg / ml); A plasma (1: 500); L plasma

  (1: 500) and IgG of H (20 mg / ml) H is a patient

  positive for PT-NANBH antibodies because it was a hemophiliac that had received a Factor VIII that had not been heat treated. At the end of the reaction, each filter was blinded to positive (when there were obviously two types of signal) or negative (when all the plates gave the same signal) It could be a

  subjective judgment and the results were therefore

  only those filters for which an acord was reached by the majority were considered

  positive The results are presented in Table 1.

TABLE 1

A & L A L H

JG 1 + +

JG 2 + + + +

JG 3 + + + +

  JG 1 appeared to react only with antibodies of patient A and not of I or H; it's not

  what would be expected of a true polypeptide

  both PT-NANBH-linked recombination and hence JG 1 was excluded from the assay. Moreover, JG 2 and JG 3 showed clear positive reactions with all three.

  PT-NANBH serums A, L and H; we continued their analysis

  is. The type of analysis described above was repeated for

  JG 2 and JG 3, except that the filters were divided

  in smaller portions and that these have been

  incubated with positive and negative serum panels

  Samples of positive sera included sera from 10 haemophiliacs and 9 intravenous drug users (IVDA). These were the best source of positive sera even though

  actual positive was unknown The sampling of

  negative rums was obtained from accredited donors closely controlled for many years by the North London Blood Transfusion Center, Deansbrook

  Road, Edgware, Middlesex, U K and have never presented

  no sign of infection for a whole series of

  including PT-NANBH The results are presented

in Tables 2 and 3.

22 -

TABLE 2

I.D.

V 19146

V 27083

V 29779

V 12561

V 15444

V 18342

V 8403

V 20001

V 21213

hemophiliacs

M 1582

M 1581

M 1575

M 1579

M 1585

M 1576

M 1580

M 1578

M 1587

MI 577

  JG 2 4/4 2/4 0/4 0/5 3/4 4/4 3/4 4/4 3/4 4/4 / 5 3/5 / 5 3/5 1/5 1/5 1 / 5 1/5 2/5 JG 3 0/5 0/5 0/5 4/5 / 5 0/5 0/5 0/5 0/5 4/5 / 5 0/5 / 5 0/5 1 / 5 0/5 0/5 3/5 1/5

The positives are underlined.

TABLE 3

IVDA JG 2 JG 3

JG 2 + JG 3

JG 2 or JG 3

6/9 (66%)

2/9 (22%)

1/9 (11%)

7/9 (77%)

Haemophiliac

/ 10 (50%)

4/10 (40%)

3/10 (30%)

6/10 (60%)

Donor

credited

0/10 (0%)

0 / Y O (0%)

0/10 (0%)

0/10 (0%)

  These data are consistent with the hypothesis that the two results of recombination ex-

  prefer polypeptides associated with a

  responsible for PT-NANBH and that these polypeptides are not identical but they can share some antigenic sites. EXAMPLE 5 Restriction Mapping and Sequencing of JG 2 and JG 3 DNA A portion (10 μl) of phage stocks of JG 2 and JG 3 was slurried to denature the phages and expose the DNA This DNA was then used as a template in a PCR amplification using Taq polymerase; each reaction contained the following in a final volume of 50 μl: Om MTris-HCl, 50 m MKC 1, 3.5 mM Mg C 12, 0.01% gelatin, pH 8.3 at 25 ° C plus IVD As oligonucleotide primers d 19 and D 20 (SEQ ID Nos. 1 and 2)

  respectively; 200 ng each); these primers are so-

  killed in the lambda sequences flanking the Eco RI cloning site and thus priming the amplification of everything

  what is cloned in this region.

  A portion of the reaction was analyzed on a gel

  agarose at 1% and compared to the markers

  JG 2 produced a fragment of about 2 Kb; The remainder of the reaction mixture was extracted with phenol / chloroform in the presence of

  m EDTA and 1% SDS and the DNA recovered by

  The amplified material was then digested with 201 μl of Eco RI for 60 minutes at 37 ° C. and separated on a 1% LGT agarose gel in TAE. The fragments decreased in size as expected and were

  eluted and purified by means of Elutips (S & S).

  serts JG 2 and JG 3 were ligated with p UC 13 digested

  by Eco RI and transformed into strand TG 1 E coli

  recombination were identified as white colonies on X-gal / L-Amp plates (L-agar plates supplemented with 10 μg / ml ampicillin,

  0.5 mg / ml X-gal) and have been verified by prepara-

  small-scale plasmid and digestion

  Eco RI reducing zymes to determine the size of

  the DNA insert The plasmid resulting from the recombinant

  its containing the insert JG 2 was called DM 415 and the one

containing JG 3, DM 416.

  The sequence of the JG 2 insert was determined by placing

  double-stranded sequence of the plasmid DNA and by sub-

  cloning into M 13 sequencing vectors such as mp 18 and mp 19 followed by single-strand sequencing The sequence of the JG 3 insert was determined in the same manner The resulting DNA and acid sequences The resultant amines are shown in SEQ ID Nos. 3 and 4. EXAMPLE 6 Expression of the PT-NANBH polypeptide in E. coli The plasmid p DM 416 (5 μg) was digested with Eco RI (20 U) in a final volume of 20 μl and the 1 Kb insert recovered by elution in a 1% LGT agarose gel. This material was then "polished" using a Klenow fragment and

  a mixture of NTP to fill the ends

  lead of EcoRI The DNA was recovered by precipitation

  ethanol extraction after extraction with phenol / chloroform The blunt-edge fragment was ligated

  in p 107 cleaved / phosphatased Sma I (a vector that allows

  clone at the 3 'end of Lake Z) and then

  transformed into TG 1 E coli cells There has been an increase

  The transformants containing the required plasmid resulting from the recombination were identified by hybridization using a radioactive probe produced by PCR amplification of the JG 3 recombinant product. were analyzed by digestion with reducing enzymes (Sal I)

  mini-plasmid preparations to determine the orientation

  One-quarter of these prescription products are

  pairing were correctly oriented to express

  the PT-NANBH sequence as a fusion with 5-

  galactosidase One of these (p DX 113) was taken

to continue the analysis.

  A colony of p DX 113 was used to inoculate 50 μl of L broth, incubated at 37 ° C. and stirred until

  the "mid-log" phase and pushed to express itself by

  After 3 hours the cells were harvested by centrifugation at 5000 g for 20 minutes.

  minutes, resuspended in 50 ml PBS and

  The pelleted cells were put back into

  suspension in 5 ml of buffer solution (25 m

  HC, 1mM EDTA, 1mg / ml lysozyme, 0.2% (v / v) Nonidet-P 40,

  p H 8.0) per gram of lozenge and incubated at 0 C

  At 2 hours, the bacterial DNA emitted was digested by the addition of D Nase I and MgSO 4 to achieve

  final concentrations of 40 μg / ml and 2 m M respectively

to reduce viscosity.

  This crude lysate was analyzed by PAGE and the model of

  Coomassie blue stained protein A protein from

  viron 150 k D was induced in p DX 113 containing

  bacteria and it has been estimated that this protein represents

  was 10-15% of the total protein Similar gels

  res were transferred to the PVDF membrane (GRI, Dun-

  mow, Essex, UK) and incubated membranes

  with positive and negative PT-NANBH sera; the pro-

  150 k D reacted with sera A and L but not with normal human serum Control traces

  containing lysate of E. coli expressing β-galacto-

  sidase did not react with the A, L or

normal human serum.

  Urea was added to the crude lysate to have a final concentration of 6M and the insoluble material was recovered by centrifugation. The 6M urea extract was used to directly cover the microtiter wells for 1 hour at 37.degree. The wells were washed three times with double distilled water and then blocked by addition of 0.25 ml BSA to

  0.2% per well containing 0.02% NaN 3 for 20 minutes.

  at 37 C Your plate was then sucked up

  Control plates coated with a crude lysate of an E. coli strand producing β-galactosidase (p XY 461) were produced in the same way. These plates were used in ELISA assays as described in FIG.

example 10.

  EXAMPLE 7 Expression of the PT-NANBH Polypeptide in Insect Cells

  The PT-NANBH insert of JG 3, isolated as described in Example

  ple 5, was cloned in chassis with the first 34 nu-

  polyhedrin cleotides in the vector p Ac 360 (Tuck-

  ow and Summers, Biotechnology, 1988, 6, 47-55), in use

  reading our knowledge of the lac Z gene reading frame in the gtll vector. Oligonucleotides were synthesized which were able to hybridize to gtll sequences flanking the Eco RI cloning site and

  which would allow amplification of the insert by PCR.

  These oligonucleotides included sites of

  Bam HI triction properly placed to allow the

  direct cloning into the Bam HI site of p Ac 360, using

  the gene inserted in the frame with the ter-

  amino acids of polyhedrin.

  A small amount of gtll construct

  JG 3 was boiled to expose the DNA and then used

  in a PCR amplification containing oligonucleotide primers d 75 and 76 (SEQ ID NO: 6), 200 mg)

and 0.5 U Taq polymerase.

  After amplification, the reaction was extracted with an equal volume of phenol / chloroform, precipitated with

  ethanol and digested with 10 U Bam HI in one

  30 μl final lipid The amplified fragment was resolved in a 1% agarose gel, eluted and ligated into Bam HI digested p Ac 360 to produce the construct.

  The plasmid resulting from the reaction is

  combination (2 μg) and wild-type Ac NPV (tug) DNA were cotransfected into insect cells by precipitation of calcium phosphate Te negative inclusion virus resulting from recombination was selected by visual selection After three rounds of plaque purification, the virus resulting from recombination (BHC-5) abounded and the expression of the

  protein resulting from recombination in cells

* the insect was evaluated by SDS-PAGE, Western blot and ELISA A protein abundantly expressed

  about 70 kD is produced in the contami-

  This protein reacts with PT-NANBH sera

next Western blot and ELISA.

  Another Bacillovirus (BHC-7) recombinant was constructed to include JG 2 sequences

  in addition to the JG 3 sequences present in BHC-5, as

  Figure 1 The PT-NANBH sequences present in JG 2 were amplified and cloned in the vector p Ac 360 as above to produce p DX 118 and the appropriate Bam HI / Sal I fragments of p DX. 119 and p DX 118 were linked together in that order in

  p Ac 360 to produce the transfer construct p DX 122.

  Plasmids resulting from recombination were identified by hybridization and orientation of the DNA

  inserted determined by analysis of reducing enzymes.

  The resulting virus from recombination was produced

  as described above and the protein expressed analy-

  by SDS-PAGE, Western blot and ELISA A poly-

  very abundant peptide (40% of total cell protein) 95 k Da reacting with PT-NANBH sera

  was found in infected cells.

  EXAMPLE 8 Purification of Pol Ypeptide DX 113

  The E. coli TGA strand containing the plasmid p DX 113 (app.

  peeled strand WDL 001) was incubated and induced in a

  1.5 liter liar (Model SET 002, SGT, Newhaven, East Sussex, UK) at 37 ° C. for 5 hours. The cells were harvested by centrifugation at 5,000 g.

  for 20 minutes and processed as follows.

a) Extraction.

  The wet cells are resuspended (1:20, w / v) in buffer solution A (50mM Tris-HCl, OmM NaCl, AmM EDTA, 5mMDTT, 10% ( v / v) glycerol, pH 8.0) Lysozyme is added to 5 mg solid per ml of suspension and the mixture is left at 4 ° C. After 15 minutes, the mixture is sonicated (6 μm peak-to-peak amplitude) over ice for a total of 3 minutes (6 x 30 sec pushes) D Nase I was added at 4 μg per ml of suspension and the mixture was left for 30 minutes The suspension was centrifuged for 20 minutes at 18,000 g (max) and the supernatant

discarded.

  The pellet was resuspended in solution

  Buffer B (25 m Hepes, 4 m M urea, 5 m M DTT, ph 8.0)

  in a ratio of 1: 6 (w / v) to obtain a suspen-

  This was centrifuged at 18,000 g (max)

  for 20 minutes and the supernatant spread out

  The sample was resuspended in buffer solution C (25 mM Hepes, 8 M urea, 2 mM DTT, pH 8.0) in a ratio of 1: 6 (w / v); before suspending,

  leupeptin (lug / mil), pep-

  statin (lug / ml) and E 64 (μg / ml). The suspension was centrifuged at 18,000 g (max) for 30 minutes and the

  supernatant decanted and preserved The lozenge was

27 -

  suspended in 25 m Hepes, 1% SDS p H 8.0.

b) Chromatography.

  The supernatant of the 8 M urea fraction was diluted 1: 5 (v / v) in 25 m Hepes, 8 M urea, 2 m M DTT, p H 8.0 and fractionated on a 7 ml column. Q-Sepharose The proteins were eluted via a 0-1 M Na CI salt gradient. Chromatography and data manipulation were monitored by FPLC (Pharmacia). DX 113 eluted at about 500 mM NaCl and was virtually homogeneous following SDS Page analysis and Western blotting. EXAMPLE 9 Purification of the BHC-5 Polypeptide Sf 9 cells (2 × 10 9) were contaminated with a stock of BHC-5 virus resulting from the recombination (moi 5). After incubation at 28 ° C. for 2 days, the

  cells were harvested by centrifugation and

continued treated as follows:

a) Extraction.

  The wet cell mass (1.2 g) was resuspended in 6 msl of buffer solution A (25 mM Hepes, mM DTT, 1 μg / ml leupeptin, 1 μg / ml pepstatin,

  Jug / mo of E 64 p H 8.0) Cells put back into suspension

  were placed on ice and sonicated by 3 shocks of 15 seconds (peak-to-peak amplitude

  um) separated by periods of rest of 30 seconds.

  The sonicated suspension was centrifuged at 18,000 g (max) for 20 minutes and the supernatant discarded. The pellet was resuspended in buffer solution A plus 4 M urea (6 ml) and centrifuged at 8000 g ( max) for 20 minutes The supernatant was discarded and the pellet re-extracted with buffer solution A plus 8 M urea (6 ml) After centrifugation at 18.00 g (max) for 30 minutes, the supernatant was retained and diluted 1: 6 in the buffer solution plus 8 M

  urea This extract was chromatographed on a

  mono-Q balanced in the same buffer solution

  column was eluted by means of a salt gradient (0-

  1 M NaCl) on 1 column volume BHC-5 eluted at about 0.45-0.55 M NaCl and had a purity greater than 90% as estimated by SDSPAGE.

was about 70%.

EXAMPLE 10

  Performance of DX 113 and BHC-5 and 7 polypeptides in an ELISA

  Microelisa plates (96 well, Nunc) were directly

  covered with 50 mm buffer solution of

  carbonate (50 m M sodium bicarbonate and 50 m M carbona

28 -

  of sodium, titrated to a pH of 9.5) with either a

  BHC-5 crude sat of 6 M urea or with p DXI 13 puri

  The plates were blocked with 0.2% BSA and then incubated for 30 minutes at 37 ° C. with sera diluted 1:20 (bacillus) or 1: 100 (E coli) after washing in saline. Tween (0.85% saline, 0.05% Tween 20, 0.01% Bronidox),

  the plates were incubated with immunoglobulins

  anti-human goat in conjunction with the peregrine

  xydase (1: 2000) for 30 minutes at 37 ° C. The plates were then washed in Tween saline and stained by adding the TMB chromogenic substrate.

  (tetramethyl-benzidine-HCl) (100ul / cup) and incubated

  for 20 minutes at room temperature.

  action was stopped with 50 μl of 2 M sulfuric acid

  and OD 450 determined (Table 4).

TABLE 4

  Indirect anti-human IQ ELISA format for the detection of NANB Bacillus E. coli BHC-5 antibodies (solid phase DXl 13 solid phase)

> 1.670

1,855 1,531

1,083 3,015

  Patients' sera 1,842 1,558 at high risk posi 0,526 0,638 tifs in the test> 2 1,516

1,823 1,602

1,779 1,318

1,122 0.616

1,686 1,441

0.259 0.205

0.158 0.120

0.298 0.209

  Serums of patients 0.194 0.111 high risk nega 0.282 0.181 tifs in test 0.263 0.165

0.184 0.163

0.121 0.099

0.243 0.104

Accredited donor 0.224 0.119

  Sera from patients at high risk of PT-infection

  NANBH (IVDA's, hemophiliacs) have been tried as

  writing; all data are expressed as O D 450 readings, with the accredited donor as the negative control In this particular group of sera, 10/19

  are positive in both solid phases-

  In addition, DX 113 was conjugated to a phosphatase

  caline by means of SATA / maleimide reduction and immunometric analysis was performed NANB sera

  positive and negative data have been diluted as

  in the serum of the accredited donor and added to

  a solid phase covered with BHC-7 Either

  after incubation (30 minutes at 37 ° C),

  added the conjugate DX 11 13 (50 l-1, 1: 2000) After incubation

  at 37 ° C. for 30 minutes, the plates were washed with a 50 mM solution of bicarbonate buffer and stained using an IQ amplification system.

  Bio and the determined OD 492 (Table 5).

TABLE 5

  Immunometric ELISA (labeled polypeptide) for the detection of NANB antibodies

Positive Negative Acquiring Donor

  in the test in the test? credited

> 0.217 0.234

0.821 0.252

> 0.214

0.542 0.257

0.876 0.308

1,583 0,278

> 0.296

> 0.273

1,830 0,262

> 0.251

  So, whether in the format-antiglobulin assay or in the immunometric assay, all high-risk patients give

concordant results.

  EXAMPLE 11 Vaccine formulation A vaccine formulation may be prepared by conventional techniques using the following components in the amounts indicated: Viral polypeptide PT-NANBH> 0.36 mg Thiomersal 0.04-0.2 mg Sodium chloride <8 , 5 mg

Water 1 mi.

-

EXAMPLE 12

  Production of monoclonal antibodies against polyps

  The DNA insert of DM 415 was subcloned into the bacillovirus transfer vector p 36 C and the resulting virus.

  both the recombination produced by a method es-

  substantially similar to that described in Example 7.

  Te virus resulting from recombination has been called

  BHC-1 and had very low levels of protein

  PT-NANBH-specific Sf-9 cells (5 × 10 7 cells / ml) contaminated with BHC-1 were lysed in PBS containing 1% (v / v) NP 40 and centrifuged at 13000 g for 2 minutes. The supernatant was passed on Extractigel-D (Pierce Chemicals) to remove the detergent and then mixed as a 1: 3 emulsion with Freund's complete adjuvant. Subcutaneous injection of 0.1 ml of emulsion (equivalent

  at 5 x 10 6 cells) to 14 and 28 day old mice followed by

  Before the injection, the mice were injected

  0.1 ml intraperitoneal (equivalent to 5 × 10 6 cells).

  l.es) of a detergent-free extract of Sf-9 cells contaminated with BHC-7; BHC-7 contains a DNA insert

  produced by ligating together the regions of

  DM 415 and DM 416 (Example 7)

  removal of the spleen three days later.

  The spleen cells were melted with N S myeloma cells in the presence of PEG 1500 using standard techniques. The resulting hybridoma cells were selected by growth in HAT medium (hypoxanthine, aminopterin, thymidine) 10-14. days

  after the fusion, the supernatant was examined in a manner

  re-selective by ELISA to detect anti-

  PT-NANBH The wells that showed reactivity to both the DX 113 and BHC-7 antigens (Example 10) were identified and the individual colonies were

  transferred to other wells, incubated and re-tested

  Cups with specific reactivity at this stage were re-cloned at a limited dilution

to ensure monoclonality.

  EXAMPLE 13 Detection of the viral nucleic acid PT-

  NANBH in seropositive patients Serums: The samples of 1400 donors enrolled in

  a prospective study on post-transfusion hepatitis

  were transfused at -20 ° C. Specimens before transfusion and after serial transfusion of the 260 vessels were stored in the same way. Transfusion samples were collected every two weeks for 3 months, once per 31 months. for 6 months and 6 months later, up to 18 months Donor and container frozen sera for three PT-NANBH events in 1981 were also available for study The diagnosis of PT-NANBH was based on an increase in -

  transferase of the amino acid alanine of the

  rum (ALT), exceeding 2.5 times the upper limit of normal in at least two separate samples after transfusion Other hepatotropic viruses were excluded by serologic testing and non-viral causes of hepatocellular injury were excluded by

  conventional clinical and laboratory studies

re.

  Immunoassay: Serum samples were tested

  retrospectively to detect the presence of antibodies against HCV (C 100 antigen)

  Ortho Diagnostics ELISA kit used according to the

  manufacturer's instructions Reactive sera at several

  several times were titrated to the end point

  in a human serum negative for anti-C 100.

  Detection of PT-NANBH viral sequences: the RNA of the

  rum or plasma was extracted, subjected to reverse transcriptase and amplified as said below The oligonucleotide primers of the reverse transcriptase / PCR were derived from the nucleotide sequence of clone JG 2

  isolated in EXEMPT 3, and synthesized on a synthetic

  Applied Biosystems 3831 A Scanners

  Oligonucleotide primers were as follows: Designation SEQ ID N Product size d 94 sense 8 829 bp d 95 antisense 9 Nonsense 10 402 bp N 2 antisense 11 (i) Extraction RNA -50 μl serum (or Plasma) was added to 200 μl by adding sterile distilled water. The 200 μl sample was added to an equal volume of buffer solution 2 × PK (2 × PK = 0 2 M Tris Cl, p H 7). 0.5mM EDTA, 0.3M NaCl, 2% w / v SDS, proteinase K 200 μg / ml),

  mixed and incubated at 37 ° C. for 40 minutes.

  The teens were removed by extracting twice with phenol / chloroform and once with chloroform only. 20 μg of glycogen was added to the aqueous phase and the RNA was then precipitated by adding 3 volumes of ice cold absolute ethanol. at -70 C 32 -

  for 1 hour, the RNA was pelletized in a centrifuge

  Eppendorf flask (15 minutes, 14000 rpm, 4 C) The pellet was washed once in 95% ethanol, dried under vacuum and dissolved in 10 ml of sterile distilled water. The RNA solutions were stored at -70 ° C. (ii) Synthesis AD Nc A mixture of 10 μl containing 2 μl of RNA solution, ng of synthetic oligonucleotide of 95, 10 m M Hepes-H Cl p H 6.9 and 0.2 This mixture of 10 μl was coated with 2 drops of mineral oil, heated for 2 minutes in a water bath at 90 ° C. and rapidly cooled on ice. of the AD Nc was carried out after adjusting the reaction so that it contained 50 mM Tris-HCl pH 7.5, 75 mM ClCl, 3 mM Mg C 12, 10 mM DTT, 0.5 mM d ATP, d CTP, d GTP and d TTP, 20 units R Nase inhibitor (Pharmacia)

  and 15 units of cloned MIV reverse transcriptase (Phar-

  macia) in a final volume of 20 μl The mixture of μl was incubated at 37 ° C. for 90 minutes.

  synthesis, the AD Nc was stored at -20 C.

(iii) "Nested" PCR

  Throughout this study, PCR results

  incorrect measures have been avoided by strict application

  measures to prevent contamination of

  Kwok and Higuchi (Nature, 1989, 339, 237-238).

  a) Tour I Polymerase chain reaction was carried out in a mixture of 50 μl containing 10 m M Tris-HCl p H 8.3, m M KC 1, 1.5 m M Mg Clz, of the gélative at 0.1% w / v, 1

  Taq DNA polymerase unit resulting from the recombinant

  (Perkin Elmer Cetus), 200 μM d NTP, 30 ng of each "outer" primer (d 94 and 95, SEQ ID Nos. 8 and 9 respectively) and 5 μl of AD Nc solution After a first denaturation 5 minutes at 94 ° C., 35 cycles of 95 ° C. for 1.2 minutes, 560 ° C. for 1 minute, 72 ° C. for 1 minute were carried out, followed by a final extension of 7 minutes at 72 ° C. (Techne PHC-1).

Automated Thermal Cycler).

  b) Turn 2 The reaction mixture was as described above

  for Round I, but 125 ng of each primer "inter-

  ## EQU1 ## and N 2 (SEQ ID No. 10 and 11 respectively) were used instead of the "outer" primers d 94 and 95. A small sample of 1 μl of the PCR products of Tour I was transferred to the reaction mixture. of 25 cycles of 95 ° C. for 1.2 minutes, 46 ° C. for 1 minute, 72 ° C. for 1 minute, followed by extension at 72 ° C. for 7 minutes. PCR products of Tour 1 and Tour 2 were analyzed by electrophoresis on a 2% agarose gel.

  Strips were visualized by staining with

  mildew of ethidium and photographed at 302 nm.

  Predictive value of anti-CI 00 and PCR serology in the prospective study: Six of 1400 donors (0.43%) enrolled in the prospective study presented antibodies against C 100 in their serum. positive antibodies, only one (donor D 6) was found to be contaminated as

  could appreciate it by the development of seroconver-

  PT-NANBH and HCV in a recipient (recipient R 6)

see table 6 below.

  The viral sequences were detected by PCR in the serum of the D 6 donor but not in the sera of the other five seropositive donors. The R 6 o recipient developed the p T-NANBH had also received blood from seven other donors (D 7 to D 13) The sera from these donors were tested and found to be negative

  for both antibodies and for PCR.

TABLE 6

SUMMARY OF DATA DONOR / RECEIVER

PROSPECTIVE STUDY

  Donator Dni D 2 D 3 D 4 D 5 D 6 D 7 D 8 D 9 D 10 D 11 D 12 D 13

DONORS

anti-HCV + + + + + +

RECIPIENT:

PCR PT-NANBH Serial Container

conversion

  anti-HCV R1 No No R 2 No No R 3 No No R 4 No No R 5 No No + R 6 Yes * Yes * * Incubation period 1 month + Seroconversion 5 months after transfusion 34 -

EXAMPLE 14

  Isolation and Expression of Additional PT-NANBH DNA Sequences The lambda gtll libraries prepared in Example 2 were also selected with the high-risk patient sera for PTNANBH but which did not react with the viral antigens, DX 113, BHC-5 and BHC7, the reasoning being that they might well contain

  antibodies that recognize different antigens.

  PJ-5 sera (The Newcastle Royal Infirmary, New-

  castle), Bi-rm-64 (Queen Elizabeth Medical Center, Bir-

  mingham), PG and Le (University College and Middlesex

  School of Medicine, London) met this challenge.

  and were used to select library

  following a procedure identical to that described

  in Examples 3 and 4 A series of elements

  resulting from recombination have therefore been identified, none of which have crossed paths with JG 2 probes and

  JG 3 One of the recombinant products BR 1 1, identical to

  by reaction with PJ-5, was chosen to

follow the analysis.

  The clone, BR 11, contained an insert of approximately 900 bp which was amplified by PCR using primers d 75 and d 76 (SEQ ID Nos. 6 and 7) as described in Example 7. was directly cloned into the bacillovirus vector p Ac 360 to form p DX 128 con-

  holding an open reading frame in phase with the first 11 amino acids of polyhedrin Bacillovirus stocks resulting from recombination (called BHC-9) were produced according to the procedure described in

  Example 7 Insect cells were contaminated

  with a virus resulting from purified recombination

  and a polypeptide of about 22 kD was obtained in

  radioactively labeled cell extracts.

  The amplified insert of B Ril was also cloned into the phage vector p UC 13 and M 13 for sequencing; DNA and amino acid sequence data are presented in SEQ ID No. 5 The insert contains 834

  bp plus Eco RI linkers added during cloning.

EXAMPLE 15

  Performance of the BHC-9 polypeptide in an ELISA

  An ELISA was established using micronutrient cups

  assays coated with an infected cell extract BHC-

  9 and an anti-Ig Ig conjugate detection system.

  following the procedure described in Example 10.

  - A variety of high-risk sera were tested in

  parallel with BHC-7 and BHC-9 and was also reviewed

  PCR method using the method described in Exem-

  The results are shown in Table 7 in which the positive samples are underlined.

TABLE 7

  PCR number BHC-7 BHC-9 l + 2.09 2.0

2 + 2.09 2.0

3 + 1.89 1.37

4 + 1.57 0.27

+ 1.26 2.00

6 + 0.91 2.0

7 0900.51

8 + 0.84 1.19

9 0.53 0.43

0.45 2.0

11 + 0.37 1.07

12 0.32 2.00

13 0.23 0.30

14 0.15 0.43

+ 0.16 0.76

16 0.09 1.74

17 0.27 2.00

18 0.15 2.00

19 0.12 2.00

0.08 0.05

* 0.27 0.29 cutoff

  Of these 20 samples, 50% are clearly posi-

  ti Es with the BHC-7 while 85% are positive with the

  BHC-9 Two samples (11 & 12) that are at the limit

  te positive with the BHC-7 are clearly positive

  with BHC-9 and some samples that are

  calf of the cut or below with the BHC-7 are positive with the BHC9 In addition, two samples (13

  & 15) that were borderline or negative with the BHC-

  7 but positive with BHC-9 are PCR positive.

  Overall there are only two samples (13 & 20) that are negative with both polypeptides and PCR.

EXAMPLE 16

  Isolation of PT-NANBH DNA sequences overlapping existing clones

  Immunological selection of expression libraries

  AD Dc described in Examples 3,4 and 14 can only identify clones that contain an immunoreactive region of the virus. Another approach to producing PT-NANBH specific clones is to use PCR to amplify the molecules of AD Nc

  that overlap existing clones Games of

  Morces can be prepared when one member of the pair is in existing cloned sequences and the other outside; this approach can be extended

  to "nested" primer pairs as well.

  AD Nc prepared as in Example 1 was amplified by PCR, with single or "nested" pairs of primers,

  using the reaction conditions described in the Exem-

  The approach is illustrated by the use of the following primer pairs: d 164 (SEA ID No. 12) and d 137 (SEQ ID No. 16); d 136 (SEQ ID NO: 14) and d 355 (SEQ ID NO: 15); d 156 (SEQ ID No. 16) and 92 (SEQ ID No. 17) A member of each pair is designed to prime in existing cloned sequences (d 137 and d 136 prime respectively in the 5 'and 3 ends) of BRII, d 92 primer the 5 'end of JG 3) The other primers

  are based on sequences available for

  agents PT-NANBH I, primer d 164 corresponds to the

  bases 10 to 33 of Figure 2 in Okamoto et al, Ja-

  Pan, J Exp Med, 1990, 60, 167-177) The primers d 155 and d 156 correspond to positions 462 to 489 and

  3315 to 3337 respectively of Figure 47 of the

  European patent application 88330922 5 One or more

  substitutions of nucleotides can be made for

  to build an Eco RI recognition site near J'ex-

  5 'end of the primers, except for d 164 o a recognition site Bg 12 was introduced; these changes

  facilitate subsequent cloning of the amplified product.

  The PCR products were digested with the appropriate reducing enzyme (s), decomposed by agarose gel electrophoresis, and bands of the expected size were excised and cloned into plasmid and bacteriophage vectors as described in US Pat. Example 5 Amplified DNA Sequences 164/137 (SEQ ID No. 38), 136/355 (SEQ ID No. 19) and 156/92 (SEQ ID NO.

  ID No. 20) are presented in the sequence listing.

  These new sequences extend the coverage of

  PT-NANBH beyond that obtained by immuno-

  selection (SEQ ID NOS 3,4 & 5) These sequences, as well as others that are in regions already

  described, can be combined into a sequence

  at the 5 'end (SEQ TD # 21) and at the end

  3 '(SEQ ID NO: 22) of the PT-NANBH genome.

EXAMPLE 17

  Fusion of different PT-NANBH antigens into a single polypeptide resulting from recombination The data presented in Table 7 indicate that while more serum antibody positive samples were detected using BHC-9 as the target antigen (17/20) rather than BHC-7 (10/20), there are some

  samples (eg, # 4) that are only positive

  with BHC-7 This image is confirmed by the continuation

  In the same way, a fusion construct was in-

  dubit using the sequence from BHC-7 'and

BHC-9.

  The sequences of BHC-7 and B Hi C-9 can be combined in a variety of ways: each sequence can be positioned all-terminal to the amino acid of the

  resulting fusion and the nature of the binding sequence

  sound can also vary Figure 2 illustrates two

  possible ways to combine these sequences.

  Appropriate restriction fragments carrying appropriate reducing enzyme sites and sequences

  linkages were generated either by PCR by means of a-

  specific or by digestion of the plasmids ex-

  The transfer vector DW1 43 consists of a Bam HI / PstI fragment of DX 122 (Figure 1, the Pst site is at position 1504 JG 2, SEQ ID No. 3) linked to the 5 'end. of the entire B Rul programming region (SEQ ID No. 7) which was amplified as a PstI / BamHl fragment using primers d 24 (SEQ ID No. 23) and d 126 (SEQ ID No. 24) ); the binding region consists of six amino acids derived from primer d 126 and residual bacteriophage lambda sequences. The DX transfer vector 136 differs from DX 143 in that the BR 11 fragment was generated using d 24 (SEQ ID No. 23) and d 132 (SEQ ID NO: 25) and thus the binding region contains five lysines. These transfer vectors were used to co-transfect insect Sf-9 cells in culture with NPV Ac DNA and Purified stocks of bacillovirus plaques resulting from recombination were produced as described in Example 7 BHC-10 was produced as a result of transfection with DX 143; BHC-11 as a result of transfection with

DX 136 -

38 -

  The polypeptides resulting from the experimental recombination

  These two viruses were analyzed by SDS-PAGE

  and Western blotting The BHC-10 produced a polyp

  tide with an apparent molecular weight of 118 k Da

  BHC-11 produced a polypeptide with a molecular weight

  The two polypeptides were

  acted with sera known to react in a single ELISA

  only with RHC-7 (eg serum A) or only

  with BHC-9 (serum B 64, example 14) Both polyps

  tides differ only in the binding sequence and this may affect either their mobility on SDS-PAGE or the way they are treated in infected cells.

EXAMPLE 18

  Performance of PT-NANBH fusion antigen in an ELISA

  An ELISA was established using micronutrient cups

  Titration coated with cell extracts infected with

  BHC-9 and an anti-human Ig conjugate according to the

  described in Example 10 Table 8 presents

  data from a comparison of the two fu-

  with the other PT-NANBH antigens resulting from recombination BHC-7 and BHC-9 as well as the protein

  resulting from the recombination HCV, C-100-3 (Ortho Dia-

  Gnostic Systems, Raritan, New Jersey) Serums are grouped by reaction pattern with BHC-7, BHC-9 and C-100-3 Group I sera react violently

  with the three antigens; Group II reacts violently

  only with BHC-7; Group III reacts violently

  only with BHC-9 and Group IV reacts

  violently with only two of the three antigens.

  eawsual aied 8 rib S 9? sal uu U, Y + Y +

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O'g <

O 'Z <

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O 'Z <

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O 'Z <

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O'Z <67 I-908

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O'Z <LS

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ú-001-D

Nlnuss 8 f Vsi L

066999 Z

  These data show that both BHC-10 and BHC-lt have a similar reactivity with these sera and, which

  is more important than the two antigenic activities

  seem to have been retained by the mergers All sera from Groups II & III which do not react

  pectively with BHC-7 or BHC-9, give a reaction

  In addition it would seem that having both antigens together gives a more sensitive assay. For example, the KT sample gives ODs of 1.57 and 0.27 with BHC-7 and BHC-9 respectively,

  while with mergers, the OD is> 2.0.

41 -

SEQ ID N: 1

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 21 BASES

  STATE IN STRENGTH: single strand TOPOLOGY: linear TYPE MOLECULE: DNA synthetics that ORGANISM SOURCE OF ORIGIN: bacteriophage lambda gtli

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 19

CHARACTERISTICS:

  from 1 to 21 bases homologous to the upstream part of the lac Z gene flanking the Eco R 1 site in bacteriophage lambda gtll PROPERTIES: initiates the synthesis of DNA from the

  phage vector in the AD Nc inserted at the Eco R site 1 -

GGTGGCGACG ACTCCTGGAG C

42 -

SEQ ID N: 2

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 21 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: bacteriophage lambda gtll

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 20

CARACTERISTIQIJES:

  from 1 to 21 bases homologous to the upstream part of the lac Z gene flanking the Eco Rl site in bacteriophage lambda gtll PROPERTIES: initiates the synthesis of DNA from the

  phage vector in the AD Nc inserted at the Eco Rl site.

TTGACACCAG ACCAACTGGT A

43 -

SEQ ID N: 3

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 1770 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULAR TYPE: AD Nc to genomic RNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE IMMEDIATE EXPERIMENTATION: JG 2 library clone

thèque AD Nc in lambda gtll

CHARACTERISTICS:

  from 1 to 1770 bp portion of the polyprotein PT-NANBH PROPERTIES: structural encoding

CAA AAT GAC TTC CCA

Gln Asn Asp Phe Pro

CGG CAT GAG ATG GGC

Arg His Glu Met Gly

GTA GTA ATC CTG GAC

Val Val Island Leu Asp

CGG GAA GTG TCC GTC

Arg Glu Val Ser Val

CCA CCA GCG ATG CCC

Pro Pro Ala Met Pro

CTG GAG TCC TGG AAG

  Leu Glu Ser Trp Lys Probably No Viral Proteins

  GAC GCT GAC CTC ATC GAG GCC AAC CTC CTG TGG

  Asp Ala Asp Leu Glu Ala Asn Leu Leu Trp t O 15

  GG GAC ACC ACC CGC GTG GAG TCA GAG AAC AAG

  Gly Asp lle Thr Arg Glu Glu Ser Glu Asn Lys

30

  TCT TTC GAC CCG CTC CGA GCG GAG GAG GAT GAG

  Ser Phe Asp Pro Leu Arg Ala Glu Gl u Asp Glu

45

  CGC GCG GAG ATC CTG CGG AAA TCC AAA AAA TTC

  Pro Ala Glu Island Leu Arg Lys Ser Lys Lys Phe

60

  GCA TGG GCA CGC CCG GAT TAC AAC CCT CCG CTG

  Ala Trp Ala Pro Arg Asp Asn As Pro Pro Leu

75 80

  GCC CCG GAC TAC GTC TCC CCA GTG GTA CAT GGG

  Ala Pro Asp Val Pro Pro Val Val His Gly

95

  TGC CCA CTG CCA ACT CCT AAG ACC CCT ACT CCA CCT CCA CGG AGA

  Cys Pro I Pro Pro Pro L Thr Thr Pro Pro Pro Pro Pro Arg

105,110

  AAG AGG ACA GTT GTT CTG ACA GAA TCC ACC GTG TCT TCT GCC CTG GCG

  Lys Arg Thr Val Teu Thr Glu Ser Thr Ser Ser Ala, Ala 1 ll 5 120 l 25 al I O d a L, n ID

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6: Serve the S erv ely

VDYOD VI) SD ODD SD ODD

te A naq dsy

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0g dsy IO

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D) YD DV Y Y D D 3 D VOD DDO

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naq ja N jq I, s D

DID DV DD 3 V DDL

  dsy u ID 9 Z 9 t DO O D) D nary D.LD s Aq ely ely Ov Y VDDO D 3 Bay 53 AD

YDD IDI

ely ely la S ely

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DDY LYY 00 DD DDL

06 t a S It {

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DDV D 3 DD DD

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3 DYD DDD VVV YDI

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O'D DDD LDOO

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ODD D D

SAD SAD

003 LJW)

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fiay all dsy

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aqd Ski

LLL IDI

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DD DDY DYDO LYVL

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n ID IA Jay U 1 D

DYDO D 03 OD DD

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LDL LVII Y 3 Y

96 E D 1 d, OLL 1 ID has Xl the jas

YDD DI DDI DODI

06 AID qaw D 30 D y the A ely u ID

DD L 3 DD DYD)

naq ati L jas

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99 O 9 VD VDD

09 ú ULO VVD 08 C ye A Ie A

19 O 10

    9 E O Id aqd A YDD 3 DML D D A SAD aqd DAD DDI DfL dsy a AL naq

V) LYVI 3 13 D

all naq

DLV MDO

the A nest

J O L OVO

SLU

ODD 9 &

09 E bv e 2 lV s Ar I N LlD

VVV DYVO

oid SÀI

DDD IDD VDD DVE

U Sy Aq I Ely aw

IVY VYY VD) O DYV

OLú s AD the A bai IA

DOL O D D ú D 3)

99 E bgy A If AID 15

DDD DD 9 YDD D V

0 where ali Jqi q) I

DIV DDV DDV

ds V D V S, -

066999 Z

  q 99 AID DDD -1 d DDD usy LV Yv 98 C Old 3.DD 46 -

  ATG ACT AGG TAC TCT GCC CCC CCC GGG GAC CCG CCC CAA CCA GAA TAC 1680

  Met Thr Arg Tyr Pro Ala Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr

545 550 555 560

  GAC CTG GAG TTG ATA ACA TCA TGC TCC TCC AAT GTG TCG GTC GCG CAC 1728

  Asp Leu Glu Leu Thr Island Ser Ser Cys Ser Ser Asn Val Ser Val Ala His

565 570 575

  GAT GCA TCT GGC AAA AGG GTA TAC TAC CTC ACC CGT GAC CCG 1770

  Asp Ala Ser Gly Lys Arg Tyr Tyr Tyr Leu Thr Arg Asp Pro

580 585 590

47 -

SEQ ID N: 4

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 1035 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULAR TYPE: AD Nc to genomic RNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE IMMEDIATE EXPERIMENTATION: JG 3 library clone

thèque AD Nc in lambda gtll

CHARACTERISTICS:

  from 1 to 1035 bp portion of the PT-NANBH polyprotein PROPERTIES: probably encodes non-structural viral proteins

  ACA GAA GTG GAT GGG GTG CGG CTG CAC AGG TAC GCT CCG GCG TGC AAA 48

  Thr Glu Val Asp Gly Val Arg Leu His Arg Tyr Ala Pro Ala Cys Lys

10 15

  CTC CTC CTA CGG GAG GAG GTC ACA TTC CAG GTC GGG CTC AAC CAA TAC 96

  Pro Leu Leu Arg Glu Glu Val Thr Gln Gln Val Gly Leu Asn Gln Tyr

25 30

  CTG GTT GGG TCG CAG CTC CCA TGC GAG CCC GAA CCG GAT GTA GCA GTG 144

  Leu Val Gly Ser Gln Pro Leu Cys Glu Glu Pro Pro Asp Val Ala Val

40 45

  CTC ACT TCC ATG CTC ACC GAC CCC TCC CAC ATC ACA GCA GAG ACG GCT 192

  Leu Thr Ser Met Leu Thr Asp Ser Ser Pro His Thr Island Ala Glu Thr Ala

55 60

  AAG CG-C AGG CTG GCC AGG GGG TCT CCC CCC TCC TTG GCC AGC TCT TCA 240

  Lys Arg Arg Ala Arg Arg Ser Gly Pro Ser Ser Ala Ser Ser Ser Ser

70 75 80

  GCT AGC AGC TTG TCT GGC CCT TCC AGG TCG GCG ACA TAC ATT ACC CAA 288

  Ala Ser Gln Teu Ser Gly Ser Ser Ser Lys Ala Thr Tyr Thr Gln Island

90 95

  AAT GAC TTC CCA GAC GCT GAC CTC ATC GAG GCC AAC CTC CTG TGG CGG 336

  Asn Asp Phe Pro Asp Asp Ala Asp Leu Glu Ala Asn Leu Leu Trp Arg

105,110

  CAT GAG ATG GGC GGG GAC ATT CGC ATT GTG GAG TCA GAG GAC AAG GTA 384

  His Glu Met Gly Gly Asp Thr Island Arg Glu Glu Ser Glu Asn Lys Val

120 125

  Svú: ot D biey UD jas ely jas Bay DOD DV;) DDY YDD 1 O DD o, DO) lasg L L IL

DDJL VZ) V DDV

QUZ

8 U 01 ID 9 D I

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Z 16 VDV O 9 L

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U 9 L VDD VOD

  09 gz a S ds V OZL DD Dt YD n 1 O ely ZL 9 D Vt) DD s Ar I Bay PZ 9 Dvy 'DI s AD AID 9 L 5 D Di DDD 9 Lt naq nar

89 Y Z S) D XL: D

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DID DIY DYY DYVD

nao s Aq ia S n I 9

DIDY DYY DDY YYD

00 ú aq L UAI the S oaw

YDY DYI DDI DIY

58 E IRA 2 q I 4 the dal

DID DDY LDI DDI

0 LZ iltd nao I old o d

DYO 1 LD DDD DDD

AlD dsy dsy the S

DDD DYD DDYO DDI

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DD YDD) IDD DD

06 L SIH IA IRA Old

IYD YID DID YDD

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VYY 9 VY DDJI YVY

* 09 t dsy nl Do nest ely

LYD D 3 Y D 9 Y 9 D 9

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DYD IDD

hid e IY the AD ss

9 DL DOL

las KID

IIDI DD 9

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DIV DDI

0 y 'a S Ul D

DDII 1 D

AID jas

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1 e A aq A

DID DDY

Ol I oad i 8 l ozd Ie A

IIDD DIID

OLI dsy oad L Yf 9 ODD Biy nari

DDD DID

  ay n Aeq YVD Dt LD gzu naq na'l ja S usy jas nerq Ry usy 9 ID i LL LDI DV DO DIAl D ODY el s AD od aq L O In ner e 1 Y AID

LDD DDIL YD D) DIY DID IDD DDO

96 Z 06 Z

  SAD 12 To A dsy n LD AI 9 Ply N 1 D DDL D 9 DI ID D) Y 9 DDT IDD DD) 9 D 9

08 Z 5 LZ

   Lg dsv las naq dsy oid dsy Al D) Oid

DYD DDY DID) VD DDD D;) D O) 0) DDD)

  the S IAJL the S nl 1) DDJ ivi DDI OYD dsy oid o d e IY

DVD IDD IDD DDD

0 úZ ja S AID agad ly V

DD Y 9 LDDLL LDD

SIZ ja S n ID igl naq

DDII D YDY DID

00 E old q I s Arq ait

IDD DDY 9 DVY IDY

Al dsy o Jd p Iy

DYI DY DDD 3 DDD)

99 t B 5 ay l V dai L IY

3 DDD YDD 9 DL YDD

091 I al I n'lD ely oad

DIY DYD DD) D DDD

  9 E 1 o Id d a a d ODDS 3 DY 9 Di LL LDI 09 g IEA MOD dsy las ÀID

DVD LDL V 03

1, i, ely ag I A E 9

DVY YDD DD) Y DDD

  SZZ s Aq aq L el V nalq DV 3 YDI D Dt D 1 LD OIZ IA Ie A aq LI 5 JI Lt) LL 3 YDY JDDY Oad oad naeq oad

IDD DD DID VDD

s Aq dl I ja S nlo

DYV DDI DDI DVD

Oad 8 o N 2 ly oad

DD 9 DI 9 ODD YDD

the A * a S IRA n ID

DID DDI DID VILY

0 ú 1 dsy nr I al I the A

DYVD DID DIY YVID

8 i -

066999 Z

49 -

SEQ ID N: 5

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 834 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear TYPE MOLECUILE: AD Nc to genomic RNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: clone BR 11 of Bible

library AD Nc in lambda gtll

CHARACTERISTICS:

  from 1 to 834 bp portion of the PT-NANBH polyprotein

PROPERTIES:

  structure probably encodes viral proteins

  AGA AAA ACC AAA CGT AAC ACC AAC CLC CGC CCA CAG GAC

  Arg Lys Thr Lys Arg Asn Thr Asn Leu Arg Pro Gln Asp

10

  CCG GGC GGT GGT CAG ATC GTT GGT GGA GTT TAC CTG TTG

  Pro Gly Gly Gly Gln Val Gly Island Gly Val Tyr Leu Leu

25

  GGC CCC AGG TTG GGT GTG GCG GCG ACT AGG AAG ACT CBT

  Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser

40 45

  CAA CCT CGT GGA AGG CGA CAA CCT ATC CCC AGC GCT CGC

  Gln Pro Arg Gly Arg Arg Gln Pro Pro Island Lys Ala Arg

55 60

  GGC AGG GCC TGG GCT CAG CCC GGG TAC CCT TGG CCC CTC

  Gly Arg Ala Trp Ala Pro Gln Gly Pro Tyr Pro Pro Leu

70 75

  GAG GGC ATG GGG TGG GCA TGG GGA CTC CTG TCA CCC CGT

  Glu Gly Met Gly Trp Ala Gly Trine Leu Leu Ser Pro Arg

90

  CCT AGT TGG GCC CCC ACT GAC CCC CG CGT AGG TCG CGT

  Pro Ser Trp Pro Gly Pro Pro Arg Pro Arg Arg Pro Arg Arg

105

  AAA GTC ATC GAT ACC CTC ACA TGC GGC TTC GCC GAC TCT

  lily Val lle Asp Thr Leu Thr Cys Gly Phe Ala Asp Ser

120 125

GTC AGG TTC

Val Arg Phe CCG Pro

CGC AGG

Arg Arg

GAG CGG TCG

Glu Arg Ser

CAG CCC GAG

Gln Pro Glu

TAT GGC AAC

Tyr Gl y Asn

GGC TCC CGG

Gly Ser Arg - AAT Asn

TTG GGT

Leu Gly

CAT GGG GTA

His Gly Val

Y 9 L?

  Od aqd the A JS AI 9 s AD 9 DD DI L ILLJ ILDL V 9 D,) tlL OLZ naq dsy AID 9 l 8 DJD IVD D 99 Z naq dsy e 12 A 89 L Dll LY Dt D: Dt el V e Iy AQL

OZL; DD O DDD DAD

n I 9 5 v Ie A

ZL 9 DVD DDD DLD

  aqi 4 L nlD a 1 A t 99 VDV DYVD DYVL To AID jas

9 L 9 DIL DDD DD:

Y 9 Z 09 Z

  A IAI W IV Ja 8 S Ja S aqd el YV el V el AID Te A nrarl DID DVL DLV LYD DXDL OXDD DDD IDD DDD DDD LLL DLD if H 6 Ey by al I Lqj, e IV '3 j 1 L otad has already been dsy s YYYDJDD YVD) V'LY YV) Y) Y) D) YY D) DY Yv YZZZZZZZZ LL NQTZ LY dd, s AD aiy jas aa S usy AID DDY D) DD a DV DLD D 9 YVLD 9 o L DDL DOD ZL DDL i VV ILDD OZZ 71 SgIZ OIZ SAD oid IPA SKD AID oj qj AL sl H qa W ar I 1 a W dsy ely TO DJ DDD DO JD D 3 9 DD Dx DYY DDY 9 LY DIY DOY DYD DDD

SOZ 00 61

  the A ali as Ja S usy eas s AD dsy USV T 4, L IA SIRH 1 TO DJI DI DDY VDI DVV DDL Df XL 1 VD DVV DDV DLD JYD DVIL

Y 81 OWI

  1 e A usy 1 y te A n ID 1 AI ely ia S e Iv oaid a'1 T 4 q L naq DID DY DDD DLD VD LYL LDD DD OD OD LLY JV Dl l S Lt OL 1,591 s Das Sas naq 'a I naq y na naq aqd 8 the LI a S aqd a S s AD Al) Oald naq 8 ZSY D DA DDDDD DJDD DJLL DAY JDDD YDD LDD XV LLJ 091 Y 51 O l Yil usy AID Jq JL elr -1 A us V Iu A AID dsy n ID noaq 'lA 5 1 Ie A Afs s TH 08 LVY DDD YDY YDD Y DY' DAD DDD DVD DYD DLD LDD DDD LDD DDD JYD 0-P G UT O ú 1 e IV nao e IV 56 y ely ely AID 6 bay nauq ja S 5 Jy 5 Iy 651 y ely a S s BH Uu DD Di 3 D DDD DOV D; DD ID DOD Do 9 LV D DD; DDD X ODD IIDD ADD DD 2 LYVD bone -

066999 Z

51 -

SEQ ID N: 6

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 31 BASES

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: bacteriophage lambda gt 13

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 75

CHARACTERISTICS: from 4 to 9 bases Bam HI site from 10 to 31 bases homologous to the upstream portion

  of the lac Z gene flanking the Eco R 1 site in the bacteriophage lambda gtII from 26 to 31 bases Eco R 1 site PROPERTIES: initiates synthesis from the phage vector in the AD Nc inserted at the Eco R 1 site and introduces a appropriate Bam Hl site for subsequent cloning into

expression vectors.

TAAGGATCCC CCGTCAGTAT CGGCGGAATT C

52 -

SEQ ID N: 7

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 30 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: bacteriophage lambda gtll

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 76

CHARACTERISTICS:

  from 4 to 9 bases BamHI site of 10 to 30 bases homologous to the downstream portion of the lac Z gene flanking the Eco RI site in bacteriophage lambda gtll PROPERTIES: primer the synthesis of DNA from the phage vector in AD Nc inserted at the Eco Rl site and introduced a Bam Hl site suitable for cloning

  subsequent expression vectors.

TATGGATCCG TAGCGACCGG CGCTCAGCTG

53 -

SEQ ID N: 8

TYPE SEQUENCE: Nucleotide

SEQUENCE LENGTH: 19 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 94

CHARACTERISTICS:

  from 1 to 19 bases homologous to the bases 914 to 932 of the sense strand of JG 2 (SEQ ID No. 31 PROPERTIES: initiates the synthesis of the DNA on the strand

  negative of the genomic RNA / DNA of PT-NANBH.

ATGGGGCAAA GGACGTCC Gq 54 -

SEQ ID N: 9

TYPE SEQUENCE: Nucleotide

SEQUENCE LENGTH: 24 BASES

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 95

CHARACTERISTICS:

  from 1 to 24 bases homologous to bases 1620 to 1643 of the antisense strand of JG 2 (SEQ ID No. 3) PROPERTIES: initiates the synthesis of DNA on the strand

  positive RNA / genomic DNA from PT-NANBH.

TACCTAGTCA TAGCCTCCGT GAAG

-

SEQ ID NO: 10

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 17 BASES

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo NI

CHARACTERISTICS:

  from 1 to 17 bases homologous to bases 1033 to 1049 of the sense strand of JG 2 (SEQ ID No. 3) PROPERTIES: initiates the synthesis of DNA on the strand

  negative of the genomic RNA / DNA of PT-NANBH.

GAGGTTTTCT GCGTCCA

56 -

SEQ ID N: 11

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 17 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear TYPE MOLECUJLE: synthetic DNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo N 2

CHARACTERISTICS:

  from 1 to 17 bases homologous to the bases 1421 to 1437 of the antisense strand of JG 2 (SEQ ID No. 3) PROPERTIES: initiates the synthesis of the DNA on the strand

  positive RNA / genomic DNA from PT-NANBH.

GCGATAGCCG CAGTTCT

57 -

SEQ ID N: 12

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 22 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 164

CHARACTERISTICS:

  from 1 to 22 bases homologous to the bases 10 to 31 of the

  sequence in Fig. 2 of Okamoto et al, Japan, J Exp.

  Med, 1990, 60, 166-167, base 22 changed from A to T to introduce a Bg 12 recognition site from 8 to 13 bases of the recognition site Bg. PROPERTIES: initiates the synthesis of DNA on the strand

  RNA / genomic DNA negative for PT-NANBH and

troduced a site Bg 12.

CCACCATAGA TCTCTCCCCT GT

58 -

SEQ ID N: 13

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 30 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo dl 37

CHARACTERISTICS:

  from 1 to 30 bases homologous to bases 154 to 183 of the negative strand of B R11 (SEQ ID No. 5); bases 174, 177 and 178 modified to introduce a recognition site

of Eco R 1.

  from 5 to 10 bases of recognition site of Eco R 1.

  PROPERTIES: initiates the synthesis of DNA on the strand

  RNA / genomic DNA positive from PT-NANBH and

  produces an Eco R 1 site for cloning.

GCGAGAATTC GGGATAGGTT GTCGCCTTCC

59 -

SEQ ID N: 14

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 27 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 136

CHARACTERISTICS:

  from 1 to 27 bases homologous to bases 672 to 698 of the positive strand of B R11 (SEQ ID No. 5); base 675 changed to G

  to introduce an Eco Rl recognition site.

  from 5 to 10 bases of Eco Rl recognition site.

  PROPERTIES: initiates the synthesis of DNA on the strand

  RNA / genomic DNA negative for PT-NANBH and

  produces an Eco Rl site for cloning.

GGGGAATTCC TCCCGCTGCT GGGTAGC

-

SEQ ID N: 15

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 28 BASES

  STATE IN STRAND: single strand TOPOLOGY: linear TYPE MOLECULE: synthetic DNA ORGANISM SOURCE OF ORIGIN: chimpanzee; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 155

CHARACTERISTICS:

  from 1 to 28 bases homologous to bases 462 to 489 of the negative strand of FIG. 47, European patent application 88310922 5; bases 483 and 485 modified to introduce an Eco Rl recognition site

  from 5 to 10 bases Eco R 1 recognition site.

  PROPERTIES: initiates the synthesis of DNA on the strand

  RNA / genomic DNA positive from PT-NANBH and

  produces an Eco Ri site for cloning.

ACGGGAATTC GACCAGGCAC CTGGGTGT

61 -

* SEQ ID N: 16

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 23 BASES

  STATE IN STRAND: single strand TOPOLOGY: linear TYPE MOLECULE: synthetic DNA ORGANISM SOURCE OF ORIGIN: chimpanzee; serum infected with non-A, non-B post-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 156

CHARACTERISTICS:

  from 1 to 23 bases homologous to bases 3315 to 3337 of

  positive strand of Figure 47, European patent application

  88310922 5; base 323 changed to C to introduce an Eco R recognition site 1

  from 4 to 9 bases Eco R 1 recognition site.

  PROPERTIES: initiates the synthesis of DNA on br

  RNA / genomic DNA negative for PT-NANBH and

  produces an Eco R 1 site for cloning.

CTTGAATTCT GGGAGGGCGT CTT

62 -

SEQ ID N: 17

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 29 BASES

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-transfusional hepatitis

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 92

CHARACTERISTICS:

  from 1 to 29 bases homologous to bases 36 to 64 of the strand

  negative of JG 2 (SEQ ID N 3); bases 57, 58 and 60 chan-

  to introduce an Eco R recognition site 1

  from 5 to 10 bases Eco Rl recognition site.

  PROPERTIES: initiates the synthesis of DNA on the strand

  RNA / genomic DNA positive from PT-NANBH and

  produces an Eco R 1 site for cloning.

CGCCGAATTC ATGCCGCCAC AGGAGGTTG

63 -

SEQ ID N: 18

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 504 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-transfusion hepatitis SOURCE IMMEDIATE EXPERIMENTATION: clone 164/137

CHARACTERISTICS:

  from 308 to 504 bp, beginning of poloyprotein

PT-NANBH.

  PROPERTIES: probably encodes viral proteins

Structural es.

  GATCACTCCC CTGTGAGGAA CTACTGTCTT CACGCAGAAA GCGTCTAGCC ATGGCGTTAG 60

  TATGAGTGTC GTGCAGCCTC CAGGACCCCC CCTCCCGGGA GAGCCATAGT GGTCTGCGGA 120

  ACCGGTGAGT ACACCGGAAT TGCCAGGACG ACCGGGTCCT TTCTTGGATT AACCCGCTCA 180

  ATGCCTGGAG ATTTGGGCGT GCCCCCGCAA GACTGCTAGC CGAGTAGTGT TGGGTCGCGA 240

  AAGGCCTTGT GGTACTGCCT GATAGGGTGC TTGCGAGTGC CCCGGGAGGT CTCGTAGACC 300

  GTGCACC ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC 349

  Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn

10

  ACC AAC CGC CGC CCA AGC GAC GTC AGC TTC GGC GGT GGT AGC ATC 397

  Thr Asn Pro Pro Arg Gln Asp Val Lys Pro Phe Gly Gly Gly Gln Ile

20 25 30

  GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 445

  Val Gly Gly Val Tyr T, had Leu Pro Arg Arg Gly Pro Arg Leu Gly Val

40 45

  CGC GCG ACT AGG AAG ACT TCC GAG CGG TCG CAA CCT CGT GGA AGG CGA 493

  Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Pro Gln Arg Gly Arg Arg

55 60

CAA CCT ATC CC 504

Gln Pro Pro Island 64 -

SEQ ID N: 19

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 1107 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-transfusion hepatitis SOURCE EXPERIMENTATION IMMEDIATE: clone 136/1355

CHARACTERISTICS:

  from 1 to 1107 bp portion of the poloyprotein PT-NANBH.

  PROPERTIES: probably encodes structural viral proteins.

TCC TCC

Ser Ser

GCC AGC

Ala Ser

GGG GCG

Gly Ala

  CGC TGC TGG GTA GCG CTC ACT CCC ACG CTC GCG GCC

  Arg Cys Trp Ala Leu Leu Thr Pro Leu Ala Ala

10

  ATC CCC ACT GCG ACA CGA ATA CGC CAC GTC TTG GAT

  Ile Pro Thr Ala Thr Ile Arg Arg His Val Asp Leu

25 30

  GCT GCC TTC TGC TCC GCT ATG GTG TAC GGG GAT CTC

  Ala Ala Phe Ser Cys Ala Met Val Val Vally Asp Leu

40 45

AAG GAC

Asp lilies

CTC GTT

Leu Val

TGC GGA

Cys Gly

  TCT GTT TTC CTC GTC TCT CAG CTG TTC ACC TTC TCG CCT CGC CGA CAT

  Ser Val Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Arg His

55 60

CAG ACG

Gln Thr

GGT CAC

Gly His

GCC CTA

Ala Leu

ATG GTG

Met Val

  GTA CAG GAC TGC AAT TGT TCA ATC TAT CCC GGC CAC GTA TCA

  Val Gln Asp Cys Asn Cys Ser Tire Island Pro Gly His Val Ser

75 S 80

  CGC ATG GCT TGG GAT ATG ATG ATG AAC TGG TCA CCT ACA GCA

  Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Ala

90 95

  GTG GTA TCG CAG CTA CTC CGG ATC CCA CAA GCT GTC GTG GAC

  Val Val Ser Gln Leu Leu Arg Island Pro Gln Ala Val Val Asp

105,110

  GCG GGG GCC CAC TGG GGA GTC GCT GCG GGC CTT GCC TAC TAT

  Ala Gly Ala Hls Trp Gly Leu Ala Gly Leu Ala Tyr Tyr

120 125

usv s AD 800 r DVV D; L OZú dil jql

096 DDL 3 D

neq naq

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aqd 6 i P 98 Dil IDD) Te A Oad

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aqa noq PZ 9 Di LL XLD e ly qu

9 L DDD ID

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00 ú 56 Z

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OLE 99 Z 09 Z

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061 981 08 11

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AD IAI

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Where 1 CIII 99 -

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66 -

  ATC GGG GGG GTC GGC AAC ATAC ACT TTG ATC TGC CCC ACG GAC TGC TTC 1056

  Ile Gly Gly Val Asn Gn Asn Thr Leu Cys Pro Thr Asp Cys Phe

340,345,350

  CGG AAG CAT CCC GAG GCC ACT ACC TAC AAA TGC GGT TCG GGG CCT TGG 1104

  Arg Lys His Glu Pro Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp

355 360 365

TTG Leu 67 -

SEQ ID N: 20

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 2043 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear TYPE MOIECUITE: synthetic DNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusion hepatitis SOURCE IMMEDIATE EXPERIMENTATION: clone 156/192

CHARACTERISTICS:

  from 1 to 2043 bp portion of the poloyprotein PT-NANBH.

  PROPERTIES: probably encodes 3 viral proteins

non-structural.

TGG GAG GGC GTC TTC ACA GGC

Trp Glu Gly Phe Thr Gly

TCC CAA ACA AAG CAG GCA GGA

Gln Thr Lys Gln Ala Gly

AGC GCT ACT GTG AGC GCT AGG

Gln Ala Thr Cys Ala Arg

CAA ATG TGG AAG TGT CTC ATA

Gln Met Trp Lily Cys Leu Ile

55

ACA CCC TTG CTG TAT AGG CTG

Thr Leu Leu Leu Tyr Arg Leu

70

ACA CAC CCC ATA AAA ACC

Thr His Pro Island Thr lys Phe

GTC GTC GTC AGC AGC ACC TGG

Glu Val Val Thr Thr Thr Trp

CTG GCT GCGTAT TGC TTG ACA

Leu Ala Ala Tire Cys Leu Thr

CTC ACC CAC

Leu Thr His

GAC AAC TTC

Asp Asn Phe

GCC CAG GCC

Ala Gln Ala

CGG CTA AAG

Arg Leu Lys

GGA GCC GTC

Gly Ala Val

ATC ATG GCA

Met Ala Island

GTG CTG GTG

Val Leu Val

ACA GGC AGC

Thr Gly Ser

GTG GAT GCC CAC TTC CTG

Asp Val Ala His Phe Leu

CCC TAC CTG GTG GCG TAC

Pro Tire Leu Val Ala Tyr

CCA CCA CCA TCA TGG GAT

Pro Pro Pro Ser Trp Asp

ACT CCT CTG CGC GGG CCA

Pro Thr Al Arg Gly Pro

CAA AAC GAG GTC ACC CTC

Gln Glu Asn Glu Val Thr

80

TGC ATG TCA GCC GAC CTG

Cys Met Ser Ala Asp Leu

GGC GGG GTC CTT GCT GCT

Gly Gly Val Ala Ala Ala

GTG GTC ATT GTG GGT AGG

Val Val Island Val Gly Arg

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SLT OLI 991

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89 -

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066999 Z

71 -

SEQ ID N: 21

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 2116 BASE PAIRS

  STRENGTH STATE: single strand TOPOLOGY: linear TYPE MOLFECUJTF: synthetic DNA ORGANISM SOURCE OF ORIGIN: human; serum infected with non-A, non-B post-transfusion SOURCE IMMEDIATE EXPERIMENTATION: contig formed by AD Nc clones from the 5 'end of the genome

CARACTERISTIQIJES:

  from 308 to 2116 bp beginning of poloyprotein PT-NANBH.

  PROPERTIES: Structural and non-structural viral proteins.

  GATCACTCCC CTGTGAGGAA CTACTGTCTT CACGCAGAAA GCGTCTAGCC ATGGCGTTAG 60

  TATGAGTGTC GTGCAGCCTC CAGGACCCCC CCTCCCGGGA GAGCCATAGT GGTCTGCGGA 120

  ACCGGTGAGT ACACCGGAAT TGCCAGGACG ACCGGGTCCT TTCTTGGATT AACCCGCTCA 180

  ATGCCTGGAG ATTTGGGCGT GCCCCCGCAA GACTGCTAGC CGAGTAGTGT TGGGTCGCGA 240

  AAGGCCTTGT GGTACTGCCT GATAGGGTGC TTGCGAGTGC CCCGGGAGGT CTCGTAGACC 300

  GTGCACC ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC 349

  Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn

10 -

  ACC AAC CGC CGC CCA AGC GAC GTC AGC TTC GGC GGT GGT AGC ATC 397

  Thr Asn Pro Pro Arg Gln Asp Val Lys Pro Phe Gly Gly Gly Gln Ile

20 25 30

  GTT GGT GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG 445

  Val Gly Gly Val Tyr Leu rl Pro Arg Arg Gly Pro Arg Teu Gly Val

40 -45

  CGC GCG ACT AGG AAG ACT TCC GAG CGG TCG CAA CCT CGT GGA AGG CGA 493

  Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Pro Gln Arg Gly Arg Arg

55 60

  CAA CCT ATC CCC AAG GCT CGC CAG CCC GAG GGC AGG GCC TGG GCT CAG 541

  Gln Pro Ile Pro Lys Ala Arg Gln Pro Glu Gly Arg Ala Trp Ala Gln

70 75

  CCC GGG TAC CCT TGG CCC CTC TAT GGC AAC GAG GGC ATG GGG TGG GCA 589

  Pro Gly Tyr Pro Pro Trp Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala

85 90

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  YTID AQD JV ds YQ 4, L at TUA TUIIA TUA Old ae S DDD 909 L m JI G D OIL DDX DD D 99 DIL DAD LDDO D 9 Y

919 OL 5 909 009

  tq L qld SAD j AL te AID s AD D A A D D ar Id Id Id Id Id Id À À À À À 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8))))))))))) DD 96 P 06 v 98 ts AD oid Ul TO Id eo T J & L Sl H dj, s AD i Kj, Oad bjy ulc dsy nal AID 68 LI f XL ODD VYD JDD VDOD DVJJ DYD DOL DDLI YL DDD DY DYD DVD DL DDDZ PL -

06655999 Z

-

SEQ ID N: 22

  TYPE SEQUENCE: Nucleotide with corresponding protein

  LENGTH SEQUENCE: 3750 BASE PAIRS

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULAR TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: human; serum infected with non-A, non-B post-transfusion SOURCE IMMEDIATE EXPERIMENTATION: contig formed by AD clones from the 3 'end of the genome

CHARACTERISTICS:

  from 1 to 3750 bp portion of the PT-NANBH poloyprotein.

  PROPERTIES: non-structural viral proteins.

  TGG GAG GGC GTC TTC ACA GGC CTC ACC GAC GTG GAT GCC CAC TTC CTG 48

  Trp Glu Gly Val Phe Thr Gly Leu Thr His Asp Val Ala His Phe Leu

10 15

  TCC CAA ACA AAG CAG GCA GGA GAC AAC TTC CCC TAC CTG GTG GCG TAC 96

  Ser Gln Thr Tys Gln Ala Gly Asn Asp Phe Pro Tyr Leu Val Ala Tyr

25 30

  CAG GCT ACT GTG TGC GCT AGG GCC CAG GCC CCA CCT CCA TCA TGG GAT 144

  Gin Ala Thr Val Cys Ala Ala Gln Ala Pro Pro Ser Trp Asp

40 45

  CAA ATG TGG AAG TGT CTC ATA CGG CTA AAG CCT ACT CTG CGC GGG CCA 192

  Gln Met Trp Lys Cys Leu Arg Island Leu Lys Pro Thr Leu Arg Gly Pro

55 60

  ACA CCC TTG CTG TAT AGG CTG GGA GCC GTC CAA AAC GAG GTC ACC CTC 240

  Thr Pro Leu Leu Tyr Arg Leu Gly Ala Gln Gln Asn Glu Val Thr Leu

70 75 80

  ACA CAC CCC ATA ACC AAA TTC ATC ATG GCA TGC ATG TCA GCC GAC CTG 288

  Thr His Pro Island Thr Lys Phe Island Met Ala Cys Met Ser Ala Asp Leu

90 95

  GAG GTC GTC ACG AGC ACC TGG GTG CTG GTG GGC GGG GTC CTT GCA GCT 336

  Glu Val Val Thr Thr Trp Val Leu Val Val Gly Val Leu Val Ala Ala

105,110

  CTG GCT GCG TAT TGC TTG ACA ACG GGC AGC GTG GTC ATT GTG GGT AGG 384

  Leu Ala Ala Tyr Cys Leu Thr Thr Gly Ser Val Val Val Gly Arg

120 125

  OZú (il E () LE 90 U nl 9 D JII ja S oaa qae nf O lf O Jas a 4 W Ie A Ari a Ld elt V Ie A nar ely 096 DYVD DDV DDL DDD DYY VYD DD t) CD OL DY IUD Dyi 9 Li DDD DLD DID ODD

Where 56 Z U 6 Z

  RID el V Im A Xt D ely R l D a Ji X l R e al l alal IRA lnal Te A sq ZU 6 a De VD Vv D VDO V 9 y DO ILI D 99 DDD O OL lve ojjg j LD D 91 D CVV ei 8 Z 08 Z 9 LZ XlO naq RIî al I Ja S RC IRA el V el V RKID el V al I CID e IV KID t 2 A

  P 98 CDD MD LD DD VIY D Y DOD ILILD IDD DDD IOD IDD LL DC) D DDD DDD YDL

  OLZ 99 Z 09 g lld 'Ely aa S RIV ely ias Old Oad o IV nlq UID el V 21 V IA daj, KID 918 DM WD VDL ILDC IIDD LOV DDD DDD LDO DIID VY DD DD DL LO D Di VDC Z ûSZ t FZ AID n a us us us us us na na na na na na 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 89 DC DC DC DC DC DC DC DC Iy Lj DggE Ov Zu 5 úZ OúZ SZZ IA -a S UIY i T 4 I aeqa ely aoj naq jas lly ai Ie 21 Oid usy Al ') od 0 ZL DILD ILDI Y DDD VDV DJL 9 D 9 DLVY C 9 LD VDJL VD LLIV C 9 D DDD LVY t CCD 9 LDD naq tql jas naq Rle 2 lv naq ai Al ulo al I AID Jas air ZL 9 CIID IDY DDJ Di LL DO) Dt) YDD Ili DYI DYD vi Yi O 9) D O DLLY

Z OOZ

  4 a SIH s R ef Y dal aqd atti, rl-D niaq 21 V f 5 Y dla, SK 1 tz 9 Li LY DVD VVV DD 9 DCLC DLM DDV Df 9 V LLD DD 3 VDD DI DYVV 06 l 981 08 L Ie A o O l el V e 12 VP 1 V n Yy) 2 YV U L C SAI * tlL e 2 Y Iq J, UIl 9 L DJDL OF DD DD SD DD DD DD DD DD DD Y DD YDD g Lt OL 1 991 ngq Iy s Rr ul D s Ar aqa UI) 1 IY naq u RID 8 ZS DID YDD y YYY OVY DMILL 9 VD DCV DDC ODD DVD o IIV V 9

091 991 091

  i, L oad naq If H a S e 1 ys KN 1 id ni qa W nid dsy aqa 08 f DVYI AIDD DID YDD DDD D DEVC YVO) DIIVY 9 YI IYD Di II oto Sú 1 nar I 12 A nli 5 jy dsy Oad 12 A a II e 1 V Old DV AKID -a S Zct DILD DC VISD L 99 V DYV DDD MLD LL L IDD ODD CCD DD C DDI 01 T aqd us V dil

DLL DVYY DI

96 f -as N LD Ie A

DDI CYV OLLC

  na Z rlaq r I DID Di LL DOD UID nest garlic

DVD CYC DII

9 tl n ID Ul D i ARI

9 YD CYD DVI

0 ú 1 na 9 ali ali

CLIO DIV D;) Y

9 L -

066999 Z

SZS OZE; SIS

  Ie A bi V dil nel ely 5 y V ae S ji L usy oad ely oad ia S oid j Lq sA D I 8 i 1 010) 003 DOL D 01 030 001 YD 33 II 3 YY Y 33 ODY 3;) Y 3 3 X 1 'JOY 30 oid AI 3 D IX j I 4 JA ely usy al I oid aqd iqj AID if H dij, qa usv 9 E 1 DDD DDD DD 3 D 3 D 3 DY 3 DD DVY DI 3 DD 33 3 Dl VD : YDD VD DD 1 DOY 3 DY 96 t, 06 Fg 06 58 ta S SD xq J, S Aq O Jd MO the A al I 6 X a 8 Ja S AXD usy Sr I the A HS 88 bl LDY DI DD 3 YD DD DDD L 3 LY DD M DO DIV Yy V DDI YD 3 08 t 'g Lfr OO Lb g 99 AID' IL all UID VIV AID s AD JOS SAD aq j uo Ulo q all AID ds 0 pt YD D 1 D DI DYD 3 DD 3 10 úL YI DDI DD DD 3 Y DYD DIVY DY ODD DYD 09 t S S Â 17 Xt O 6 lD dd the A MO s K d Y AMO 6 by ul SAD ja S ag in 8 ld oad the AZ 6 ú MY MY MY MY D D 3 3 3 3 3 3 3 3 3 3 3 3 3 Y 3 3 3 3 3 3 3 3 3 3 g g g g g g g g g g g g g g g g g g g g g g g g g g g 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 q q q dsy f út YODD DDD 03 VII YD D 33 D 3 JD D 3 LD D 31 DO YD DAD DOU DD 3 D 1 Da LL DY ely narq e A j Lq I s AD a I I dj dsy daj IPA dsy 65 y nar daj a S AID

  96 ZI DD O D 3 L LL D Y DD 31 YLV DD DYD DD O YD YD DDV YL; DD O O DDI D 3 DD

  jass SD oa j 1 q L ae S s AD dsy nid usy al I dal ul D s TH naq D 6 ay s Aq 8 T MI DO DD D 33 D DODV DD 3 O DY 3 DD DVI LLY DD O DVD Dy D DAD O OV OO Ob 96 b 06 E 85 nr Iq naq u ID q I al I 1 qj naq ds j 8 ag naq a II u ID Jqt L Ie A 65 J e IV OOI Dl, D 3 DY 1 D 3 DD DAY úD 3 Y LJD DYD D 3 DAD DLY D 3 YD DY DOD ADD YDD

08 º YLE O LV

  Lly PIY dsy 'Os ft 11 o ld TA 1, I S 8 f H -It I, O d I S 12 A STH usy Ai D

  ZI 51 DD DD 3 D 3 D DD 3 OYVD 1 DD O DD LYL DYD DD 3 DDD 3 D 3 LLLD LúVD DVY LDD

Y 9 E 09 E S

  -l S ely aqd ely 811 l nar 6 J usy -8 di L u 'l 1 e A ely AID hlo 0 ol DD ODOD DDD O DLI DD y 3 VI DAD DDD DDO YES YD DIU úDD O Dol DYD AID O Od AID 1 e A s TH DXY Ba 1 y naq al I 8 IY EI s AD Te AT AT ATIA

  9501 DDD YDD 100 DIU D 3 YD DDD 1 DD D 3 D YLY DDD YD 3 D DO 01 D DD DDD D 3 D

  ú: z z z z z z z z z Il Il Il Il Il Il Il Il Il Il Il Il Il Il Il Il Il Il Il

  8001 DID D 3 DD DDD ADD 3 DD DLL D 3 D DIV DDD 30 DD DD YLL D 3 1001 D 3 D D 3 D

LL -

06655999 Z

  OZL SL Ol L -0 OL s Aq 6 W nazi ali nyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyh ,, OVO Dy IVY Day nol o Jd

ZITZ DD Y Y 1 D DAD DD 33

  n ID the A 5 i aqj 14, t 909 DYD DD DDD O DDY 0 L 9 tiy ni D al I naq

910 Z DDD 30 DY) DVDJ 3

  9 IV srq -the 8961 X) t O DYV 'JJ DD 3

00 L 969

  dsv atd ia S dsy nlq al I ITA Ie A DYO DM 3 LD DY 3 DLD D V 'Vf V D al I ds A; i D XIT fi nc h i SH iay

MLY DYD O DD DD O LY OYD 1 VD 003

  dsy e Iy dsy oid aqd dsy usy ul D DY 1 3 DD DY 3 DD DLL DY O L IV D

099 99

  Oad el as naq url las ely las IDD O DDú 3 L DJI L m DYD DY LDO ëD s Aq usy N LD

01 V DIV 010

sheepfold

1 D 3 L

9 L 9 da L nar naq usy

00 O LDD DLD) D 3 V

  aq, in II AL a It IL 3 DV Lv DI: D the S jas el naq

LDLL DOY DDD DLL

  01 '9 f 9 f O ú 9 SZ 9 -a S oad oad the S XAL Ba' ely 'the Jl 5 By 51 ys A Xq e TJ 14, The tl L> Iy 1 qil OZ 6 t DDC DDD DDD ú 31 DO OD OY 3) 3 DID) DV D-DD D v 3 DD D 3 V DYD 3 DD Y 1 DY

0 09 919

  al I s TH las oad dsy q I neq qa the S aqj, naq the A el I ZL 81 DLV DYD DD 3 DDD DY 'DDY D 1 D ol Y 3 DD 3 3 D DLD Di L YD) 9

909,009

  nlh oid nl D SAD oad neoq u LD Ja S Ai A naq Aa, ull gz 8 'I t 3 D D O DD 1 D DO 1 DD D O D 03 D DD DOD O L O 9 ID D 31 YYD

069 989 089

  lPA u 19 a 9 qj iqt, the A nie 9 nl D bj V noq naq od s Aqr SAD 9 LLI DID O YD D 01 YDL D 3 D DY L 00 YD DV D Y O D DV D úDD YYY D 30

9 LS O LS 999

  À f f f 5:::::::::::::::::::::::::::::::::: Oud

YV 9) ú O DDD

us V naq t ID

DY DLD DD 9

e IY oad e IY

DD 9 DDD IDD

0333 ú 30

nl D olad ly

VD DD 3 D 3 DD

  09 O t Oad 1 e AD o d s AD s s s s s s s s s s s s s s s s s s s s s s s s A A A A A A A A A A A A A A A A A A A M Y D Y D Y D Y D Y D Y D Y D Y D Y D Y D Y D Y Y D Y D Y D Y DD

OS 59 ú 9 O

  jL If H aqd dsy Rtl te A BJV Jq I TEA n ID Ie A LL Nlid ely el g ZU 9 ID 3 DOYD Di L 10 DOD DLD 00 DD OY LID DY 9 O LD D 3) OY 1 YJ 10 ú 30 ú O 8 L -

066999 Z

* 016 906 006

  s Aq ul D Say ul D Uas PI las ba V ias IQQ aq I PIV a KA ITA q GW usy 9 LZO DCV CV DDC DYVD DOV VDC DOV DC 3 DD VDV DDV D 13 O DVI DLC Di V DVY

968 068 988

  Sl H if H 5 a naq I narq 1 S us jas narl eel 2 s V OLI O d nor I SA I aog S 889 Z DYD DVD AO SD DLD DAL 1 i Dilk DDY DC L CLJ DYJ DA DVDD DV; D DVV DDV

088 9 L 8 O L 8 998

  nli nl OP Iy ey s AD od a Til a-EI naol e 2 V ALD tq L, dij L iq L Sj, ia S Op 9 z YYD OYVO CD DD 1 DD, YVDD DDV DZV DID 1 DO DOD YDV DD Oi VDV D 3 YI DD% aw ja S s D s AD Te A IPA dsy n ID AID

  Z 69 SZ DIY DL ODI 011 DA 10:) YD OVYO 10

e D hi V n IV 1 as

DDD 9 Y DYO LDY

the A DDV IJS

DOD DOY AD &

958 O 08

  da 11 ja S AI dsy ja S naq dsy Oid dsy p g O DO 1 D 1 ODO DYV D 9 Y DAD VY DDD Y Yv AID Oid nl D AID 1

999 DDD 9 D D 99

  0 e 8 O Id O o N Jas 96 t 9 E DDD DIY DD) SZ 8 as a Al -a S nl the TA ds V DDI L O i O DD 9 YD D DVYD 0 08 a S Alo e IV AMI

1 D 1 YOD YDD Y 90

  t 8 o 018 Ua S O O d U D d o d o d o d e Iy tt Iy 8 pgt D D DDD VVD D 9; D) i DDD DDV YDD jq L AECD as dsy

DDV DCC D 9 Y DYV

IUA Ul V ja S

DID DDC DDL

  96 L 06 L o.d n-ED Jas ja S A lth e V i s I l L a n d ly LC l Ly narl e YV ags

  OO 000 11 D 11 DDJ DOV ID D 311 IDD D 11 1 DY DDC J ID DYD DCD D 1 D D 3 D IDI 3

08 L SLL OLL

  -a S ITA Iqt L Jas nl D at 11 noql IEA ITA "TI 4 5 Ba s Aiq f 5 l B 5 Y Oad Old Zgi ALDD DD DD DD 11 VYC VDV DAD j 1 I LD V Dv DCCV DVV SD V ODCD VDD IDD

99 L 09 L 99 L

  Old al IO Id ojd q 1 t SA Kq I lq O Id oad nar O Cd s AD AID SFH -e A t O úz YDD;) Y 'DD A 1 DD DDV DVI 1 DV; DD VDD DLD YDD DCA CCDD 1 YD VIAD DD 1 OSL 9 i L Of L Oid oad TUA x A 1.1 dsy oad p 1 Y s Aq dj, 1 a S n naq naq oid oid usid 99 VDD 1 DD D 1 DY 3 D 3 YD DDD DDD 1 YV CD Di DD 1 DYOD DCD DCD CDD IDD DVV

9 úL OL 9 ZL

  A 1 A dy Oid a Vy ely dil ely oid 1 o W ely Oid oid aqd s Aq s Aq jas g ZZ DI CYD DDD DODD I O DD DD YVDO DDD OV DDD CCDD VDD DM VVV CV VY DI 3 6 L -

066999 Z

  9 ú 8 n ID naq 0 VW Ji D oi Ed DDD DOD dsy dsy

DVD DYD

0011 9601 0601

  s KD ul D J & L a n d i n d i n d i n d i n d i n d i n d i n d i n d i LDI YD j YDVX OYD DYD VJI DD DAY DY 9 LIVY DYD LDY DD Y) DY

9801 0801 GLOI

  Ias dsy aqd s AD B 6 y iq L dsy TAI 1 ely aqd AK If I od jqj L s A Kr I s Kq p 9 zg YVD DVYO ALL LDL: D D DY 3 LVYA YDO JMLL D 9 D DLY ADD DDY VYY YDIV VI

OLOI 9901 090 L

  JS s Ai d LL eiy us V ie A neaq aqd n ID on A 6 ay UID A'lO Oxd jas XAL 91 ZE YVDL y Y DDL DDD Dy V DAD DLLD XDLL VM DYD D) YO'D DYD) ID DD LDI L Ly J 9901 0901 p O i Ui D a Tid KI ÜKL -l S -as alf lew In A ely Ul D old naq uqt L las the A 891 t DY ODD Lvl DDL DDI DDD DAV DOLY JDD 9 VD O DD DL :) DD) YD:) D:) LDD ot OO 5 ú O l UOOl 5 ZUL Ie A dsy & i, wi ng Ki n I s KD) A 61 Vy Ie A KID nar dsy O Ld oi Zi DD DAD D DAY DV Y YL DL) DV V DOL O) D DOL DODO DOLLL DYD YDD OZOI 9101 Ot O 1 mud the A al I n Ga B Jy el V ola s Ki 6 Bay AIV AID By nlh Oad Ul D ILA ZLOU DLL DL D ALY LAD LID L :) VDD) YV DDD O DDD DV Df) IV DD '1 V: D DLD

9001 0001 966

  s KD aqd the A ni D US 's Kq ely Vaw al I qt aq IL dsy a Ir oad aq LL nli t O C DDL DLL Li LD DYD LVI y I YDD DVY DDY DDY DYD IAY YDD YDY VID)

066 986 086

  Xq I dsy n ID naq naq dsy n ID di L IPA la S fiay e II if H usy al I 2 y

  9 L 6 Z LDY DYVD YVD DML A DY 3 Y DDL DD DDIL DD) DLY DYD) DYV LLYV DDD

9 L 6 O L 6 996

  s Krj ja S aa S naq usy 6 bay the A dsy s Aq ely KD a KL A-lD aqd s Ki mas 8 Z 6 Z Dy JY DDY DDI VD D 1 DD; YOI DY Y y DD DD LYVI D Dt) Jil LY 11 YL: DL

096 996 096 SP 6

  s Kq ely a S S f H oid oad a Tq I naq s Aq s D -ely njl) tif 1 A ri; 'l 088 Z YVY DD) DDL YD YDD DD DDY 3 L: D Vy;: VD YY V 3 D DYD YLV YVDI'V VLD 0176 9 iu 6 0 C 6 nar skil e-Vy s Aq the A q L las ely À el 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

SZ 6 OZ 6 916

  dsy ul D ki L s TH dsy dsy naq al I ul naq fay dsy aqd xqj the A s Aq

  SLE DVYD DYD DVL DYD LYD DYD DLD DIV YYD DLD YVOY DYD DYD LLJL DDY DID VYI

08 - 066899 z OGUI oad dsv

OSLú DD DVO

  9 t, 91 OPZI 5 EZI 61 v q I naq f & L J Ai L Te A 6 V s Aq AID A S ely dsy s TH ely the A Ua S

  LV LL) D DDL DILD DVL DI V L DY VVY DO LDL D DYD J D DYD CDC DID ODL

  OV Zt SZZI OZZI Ie Ausy the S lae S s AD a S Iq & ll naq n I naq dsy j Al nl I Oid ul) 969 e DCD LVY DDL DDL DDL V 3 DL YDY VL DYVO DLL DLD DY DVL VVO VDD 'VD SIZI l U Ct S Olf L old Oad dsy AID ozd Oid e Iy a S io & 5j Ljq e V eliy ni jq L aqd 8 t 9 ú D) D) DD DDD DDD DDD DD '; DL Y Y L CDDY LDY DOLV LD O O O DDO DLL O OZT 56 t I 0611 98 t 1 Te A 6 Ji nael ae SL ye Ty dsy nl D ul D itt L Al De fl Jas nl D s KD 81 I 009 E DLD Y-0 DV LD DD DDC DDD DYD DYD DYD DDV and OD DDD DOV DOV LDL DLV 0811 SL 11 O L I L A nsq dsy dsy AID s a D i n d I s Aq Z Sgú LD D 3 DL DCD DYV YDO DOD VY DIY; :) 3 LVDDO YD DYDV DLD OVV

9911 0911 9911

  e Iy ely 5 jy s AD e IT p 1 ya S el s Aq nlq gi A s AD, jq I naq jq L usy t O OSú Yv DO IDD v OD 101 DOD YOD LDL DDD l OVY DOL D;: Y JDI YDY DLD DD V LVV

0911 O 1'0 '11

  AID s K d e S q 1 11 q nfaq IA ID a S ely IV 61 Y SAD 61 Vy 6 IV al Al D 99 t DD DD 3 L DDV IVDY DDY DID DOD DCD DDY DDD DDD DC DD COD Y)) 0 DDD SAD usy Ul D Al D s Aq a S usy a 1 q 4 naq oad AID ATID II Aj naq Day os DD DV DVD YDD Y VVV VDLV L;: VY DDD j D c 030 DD; DD Y LYV LL: D ODD OZII 591 l 0111 5011 nli D q L nnel a 8 S 51 y al I ely ul D 6 jy ely nl oad e IV naf I dsy SAD 09 úúC CDV YVDV D DLDI DDV v YV DDD DYD V 30) D VD VD DDD DDD DAAI DVO 101 18 -

06655999 Z

82 -

SEQ ID N: 23

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 23 BASES

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: Autographa californica baculovirus Nuclear Polydedrosis virus (Ac NPV)

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 24

CHARACTERISTICS:

  from 1 to 23 bases homologous to the portion of poly-

  hedrin Ac NPV downstream of the Bam Hl cloning site in

  the vectors p Ac 360 and the like.

  PROPERTIES: initiates synthesis of DNA from baculovirus transfer vector sequences which

  flank the DNA inserted at the Bam HI site.

CGGGTTTAAC ATTACGGATT TCC

83 -

SEQ ID N: 24

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 31 BASES

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA ORIGINAL SOURCE ORGANISM: Autographa californica baculovirus Nuclear Polydedrosis virus (Ac NPV)

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 126

CHARACTERISTICS:

  from 1 to 31 bases homologous to the upstream junction sequences produced when the D75 amplified AD nc (SEQ ID No. 5) is cloned into the Bam HI cloning site in p 360 vectors and the like; imbalances at the level of the bases 13 and 14 introduce a PstI site of 3 to 10 bases homologous to the Bam HI site region in p 360 and similar vectors of 4 to 9 bases Bam HI site

from 12 to 17 bases Pstl site.

  PROPERTIES: initiates DNA synthesis at the junction of baculovirus transfer vector sequences and sequences previously amplified by oligo d 75; introduced a Pstl recognition site for the

subsequent cloning work.

  TAAGGATCCC CCT GCA GTA TCG GCG GAA TTC 31

Ser Ala Ser Val Ala Glu Phe 84 -

SEQ ID N: 25

TYPE SEQUENCE: Nucleotide

LENGTH SEQUENCE: 45 BASES

  STRENGTH STATE: single-strand TOPOLOGY: linear MOLECULE TYPE: synthetic DNA

ORGANISM SOURCE OF ORIGIN: N / A

  SOURCE EXPERIMENTATION IMMEDIATE: oligomeric synthesizer

nucleotide; oligo d 132

CARACTERISTTQIUES:

  from 5 to 10 bases PstI recognition site from 13 to 27 bases binding coding for five Lys residues from 28 to 45 homologous bases alux bases 4 to 21 of B R 11

(SEQ ID 7).

  PROPERTIES: initiates DNA synthesis at the BRII end and introduces a synthetic sequence that encodes lysines as well as a PstI recognition site

  for subsequent cloning work.

  CTGCCTGCA GTA AAG AAG AAG AAG AAG AAA ACC AAA CGT AAC ACC A 45

  Val Lys Lily Lys Lily Lys Lily Lys Thr Lys Arg Asn Leu

10

-

Claims (15)

claims:   A PT-NANBH viral polypeptide comprising an antiserum   gene having an amino acid sequence which is at   less 90% homologous with the acid sequence a-   mine of which question in SEQ ID No. 3,4,5,18,19,20,   21 or 22, or is an antigenic fragment.   A PT-NANBH viral polypeptide according to claim 1 in which   which amino acid sequence is at least   90% mologue with the amino acid sequence referred to in SEQ ID No. 3,4 or 5 or is a antigenic fragment.   A PT-NANBH viral polypeptide according to claim 2, in which   which the amino acid sequence is at least   90% mologist with the amino acid sequence of which   question in SEQ ID NO: 3 or 4 or is a fragment thereof antigenic   A PT-NANBH viral polypeptide according to claim 2, wherein   which the amino acid sequence is at least   90% mologue with the amino acid sequence   qestion to SEQ ID No. 5 or is a fragment thereof   tigénique. A PT-NANBH viral polypeptide according to one of   preceding claims, wherein the   amino acid is at least 95% homologous to the amino acid sequence   SEQ ID Nr. 3,4,5,18,19,20, -21 or 22, or is a fragment thereof antigenic   A PT-NANBH viral polypeptide according to claim 5, wherein the amino acid sequence is at least 98% homologous to the amino acid sequence of SEQ ID NO: 3,4,5,19,19 , -20,21 or 22, or is an antigenic fragment.   A PT-NANBH viral polypeptide comprising an antiserum   gene from the programming region   structure of the viral genome and an antigen   from the unstructured programming area of the viral genome.   A PT-NANBH viral polypeptide according to claim 7, wherein the antigen of the structured programming region has an amino acid sequence which is at least   less 90% homologous with the acid sequence a-   SEQ ID NO: 5, or is an antigenic fragment thereof, and the antigen of the unstructured programming region has an amino acid sequence that is at least 90% homologous to the amino acid sequence. of which question to the 86 -   SEQ ID No. 3 or 4, or is an antigenic fragment thereof   than. A DNA sequence encoding a viral polypeptide   PT-NANBH according to one of Claims 1 to 8.   A DNA sequence according to claim 9 as set forth in SEQ ID NO: 3,4,5, 18, 19, 20, 21, or 22.   11 An expression vector containing a sequence   DNA according to one of claims 9   or 10, and capable in the presence of an appropriate host   to express the DNA sequence to produce a viral polypeptide PT-NANBH.   12 A host cell transformed with a vector of ex-   pressure according to claim 11.   A process for the preparation of a PT-NANBH viral polypeptide comprising cloning or syn   thesis of a DNA sequence encoding a polyp   viral PT-NANBH in accordance with one of the   1 to 8, the insertion of the DNA sequence into an expression vector such as   may, in the presence of an appropriate host, express   sea and transform a host cell by means of   vector of expression, to cultivate the cell   lule transformed host and isolate the polypeptide viral.   A polyclonal or monoclonal antibody against a PTNANBH viral polypeptide, in accordance with one of Claims 1 to 6.   A method for detecting PT-NANBH viral nucleic acid comprising: i.) Hybridizing the viral RNA present in a sample, or the AD Nc synthesized from this RNA, with a corresponding DNA sequence SEQ ID NO: 3,4,5,18,19,20,21 or 22, and the selection of the resulting nucleic acid hybrids to identify any PT-NANBH viral nucleic acid;   ii) the synthesis of AD Nc from the RNA vi   present in a sample, the amplification   preselected DNA sequence   responding to a subsequence of SEQ ID NO   3, 4, 5, 18, 19, 20, 21 or 22 and the identifica-   of the preselected DNA sequence.   87 - 16 A test kit for the detection of PT-NANBH viral nucleic acid comprising: i) a pair of oligonucleotide primers, one of which corresponds to a portion of the nucleotide sequence of SEQ ID NO 3,4, 5,18,19,20, 21 or 22 and the other of which is on the 3 'side of the first and corresponds to a portion of the complementary sequence, the pair defining   between them a preselected DNA sequence   born; ii) a reverse transcriptase enzyme for the   synthesis of the AD Nc from the RNA of the   sample upstream of the primer corresponding to the nucleotide sequence complementary to SEQ ID NO: 3,4,5,18,19,20,21 or 22; iii) an enzyme capable of amplifying the preselected DNA sequence; and optional   iv) bleach solutions and reaction buffers   tion. 2017 A method for the detection of PT-NANBH viral antigen or viral antibody, including the   contacting a sample with a polyp   viral PT-NANBH in accordance with one of the   claims 1 to 8, or a polyclonal or monoclonal antibody according to claim 14, and   the determination of the presence or absence of a link   its antigen-antibody within the sample.   18 A test kit for the detection of viral antigen   PT-NANBH or viral antibody comprising a poly-   PT-NANBH viral peptide according to one of the   Claims 1 to 8, or a polyclonal antibody   or monoclonal method according to claim 14, and means for determining the existence or not of a   antigen-antibody binding within the sample lon.   A vaccine formulation comprising a polypeptide   viral PT-NANBH in accordance with one of the   dications t to 8, in association with-a support   acceptable from a pharmaceutical point of view.